1Veterans Affairs Ann Arbor Healthcare System, Geriatric Research Education Clinical Center, Ann Arbor 48105; 2Department of Internal Medicine, University of Michigan Healthcare System, Ann Arbor 48109; and 3Department of Pathology, Wayne State University, Detroit, Michigan 48201
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hall, Karen E.,
Jackie Liu,
Anders A. F. Sima, and
John W. Wiley.
Impaired Inhibitory G-Protein Function Contributes to Increased
Calcium Currents in Rats With Diabetic Neuropathy.
J. Neurophysiol. 86: 760-770, 2001.
There is a growing
body of evidence that sensory neuropathy in diabetes is associated with
abnormal calcium signaling in dorsal root ganglion (DRG) neurons.
Enhanced influx of calcium via multiple high-threshold calcium currents
is present in sensory neurons of several models of diabetes mellitus,
including the spontaneously diabetic BioBred/Worchester (BB/W) rat and
the chemical streptozotocin (STZ)-induced rat. We believe that abnormal
calcium signaling in diabetes has pathologic significance as elevation
of calcium influx and cytosolic calcium release has been implicated in
other neurodegenerative conditions characterized by neuronal
dysfunction and death. Using electrophysiologic and pharmacologic
techniques, the present study provides evidence that significant
impairment of G-protein-coupled modulation of calcium channel function
may underlie the enhanced calcium entry in diabetes. N- and P-type voltage-activated, high-threshold calcium channels in DRGs are coupled
to µ opiate receptors via inhibitory Go-type G
proteins. The responsiveness of this receptor coupled model was tested
in dorsal root ganglion (DRG) neurons from spontaneously-diabetic BB/W
rats, and streptozotocin-induced (STZ) diabetic rats. Intracellular dialysis with GTPS decreased calcium current amplitude in diabetic BB/W DRG neurons compared with those of age-matched, nondiabetic controls, suggesting that inhibitory G-protein activity was diminished in diabetes, resulting in larger calcium currents. Facilitation of
calcium current density (IDCa) by
large-amplitude depolarizing prepulses (proposed to transiently
inactivate G proteins), was significantly less effective in neurons
from BB/W and STZ-induced diabetic DRGs. Facilitation was enhanced by
intracellular dialysis with GTP
S, decreased by pertussis toxin, and
abolished by GDP
S within 5 min. Direct measurement of GTPase
activity using opiate-mediated GTP
[35S]
binding, confirmed that G-protein activity was significantly diminished
in STZ-induced diabetic neurons compared with age-matched nondiabetic
controls. Diabetes did not alter the level of expression of µ opiate
receptors and G-protein
subunits. These studies indicate that
impaired regulation of calcium channels by G proteins is an important
mechanism contributing to enhanced calcium influx in diabetes.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Considerable evidence suggests
that impaired regulation of calcium currents in sensory neurons may
contribute to neuronal dysfunction and injury in diabetes mellitus.
Various pathological lesions are observed in animal and human models of
diabetes: segmental demyelination; atrophy; loss of myelinated and
unmyelinated fibers; Wallerian degeneration; segmental and paranodal
demyelination; and blunted nerve fiber regeneration (Greene et
al. 1992; Ozaki et al. 1996
). A variety of
pathogenic mechanisms have been described (Greene et al.
1992
), including vascular insufficiency, glycosylation of
cellular components, formation of sorbitol and other polyols, mitochondrial dysfunction (Kostyuk et al. 1999
), and
abnormal calcium signaling in peripheral sensory pathways (Hall
et al. 1995b
; Voitenko et al. 2000
). Impaired
calcium regulation in diabetes may be of particular importance as it
has been correlated with both impaired neuronal conduction and neuronal
injury. Elevation of calcium currents observed in peripheral and spinal
sensory neurons in rodent models of diabetes results in increased
cytosolic calcium release from internal stores and impaired calcium
re-sequestration (Hall et al. 1996
; Voitenko et
al. 1999
). The latter is of particular interest as prolonged
elevation of cytosolic calcium has been implicated in the pathogenesis
of neuronal injury in a variety of neurodegenerative disorders
(Orrenius and Nicotera 1994
). The observation that
diabetes was associated with increased current amplitude in multiple
voltage-dependent calcium currents (Hall et al. 1995b
;
Voitenko et al. 2000
) suggested that the underlying impairment might affect signal transduction pathways coupled to multiple calcium channel subtypes.
Neuropathic pain and sensory paresthesias are a common complication of
diabetes mellitus and are often difficult to treat with opiates. In
sensory dorsal root ganglion (DRG) neurons, k and m opiate receptors
inhibit multiple high-threshold calcium channel subtypes via a pathway
coupled primarily to the pertussis toxin (PTX)-sensitive, inhibitory
guanine nucleotide binding (G) protein, Go
(Moises et al. 1994; Wiley et al. 1997
).
We previously observed that opiate-mediated inhibition of calcium
currents was significantly impaired in a rat model of Type I diabetes
(Hall et al. 1996
); however, the underlying mechanism
for this significant diminution in opiate responsiveness in diabetes
was unclear. Using electrophysiologic and pharmacologic techniques, the
present study was undertaken to determine whether G-protein activity
and/or expression was affected by diabetes. Prepulse facilitation of ionic currents by a large-amplitude depolarizing voltage step results
in augmented current amplitude elicited by a subsequent test pulse. The
response is both time- and voltage-dependent with rapid reversal
observed within 500 ms (Ikeda 1991
). The mechanism is
thought to involve transient inactivation of inhibitory G proteins associated with calcium channels (De Waard et al. 1997
;
Ikeda 1991
). The phenomenon of facilitation is not
limited to reversal of inhibition by agonists known to act via
inhibitory G proteins but can also be demonstrated to occur in the
absence of exogenous inhibitory ligands (Doupnik and Pun
1994
). The latter suggests that "tonic" inhibition of
calcium channels occurs. Such tonic inhibition may be due to
autocrine/paracrine stimulation of receptors or may involve mechanisms
independent of receptor stimulation. Facilitation may have a
physiologic role in excitable tissues receiving convergent inputs
(Dolphin 1996
), as neuronal excitatory postsynaptic
potentials may be increased up to 50% by stimulation with repetitive
or simultaneous inputs.
Two animal models of Type 1 diabetes were investigated: the spontaneous
BioBred/Worchester (BB/W) rat and the streptozotocin (STZ)-induced
diabetic rat. This was done to determine if the alterations in
electrophysiologic and metabolic function were a ubiquitous feature of
many models of diabetes or were model-specific. At the ages and
duration of diabetes studied in this report, autonomic and sensory
neuropathy have been documented in both models. Both BB/W and
STZ-induced diabetic animals demonstrate slowing of action potentials
as measured by nerve-conduction studies. The underlying pathophysiology
appears to involve both a reversible component and an irreversible
component. The latter is likely due to neuronal loss from irreversible
nodal degeneration and distal dying back of sensory neurons, while the
former can be reversed by treatment with a variety of agents, including
insulin, other hypoglycemic agents, aldose reductase inhibitors
(Hall et al. 1995b, 1996
), mitochondrial
stabilizers, and antioxidants. The beneficial effects of agents such as
aldose reductase inhibitors on nerve conduction are independent of
blood glucose and are strongly correlated with normalization of calcium
signaling (Hall et al. 1995b
, 1996
). This was of
particular interest as prolonged elevation of cytosolic calcium has
been implicated in several models of neuronal death, thus providing a
mechanism to explain the irreversible neuronal damage observed in
chronic diabetes. However, our prior studies also documented a
reversible impairment in calcium channel modulation in diabetes.
G-protein-mediated modulation of calcium channels is a well-known
mechanism regulating calcium influx in DRG neurons. In these neurons,
activation of G proteins by ligands such as norepinephrine and opiates
is predominantly inhibitory and mediated via
Go-type G proteins (Moises et al.
1995
). Tonic activation of inhibitory G proteins has also been
documented in DRG neurons. This study was designed to determine whether
the underlying mechanism causing elevated calcium currents in diabetic
neuropathy might be diminished inhibitory G-protein activity.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animal models
Prior approval for these experiments was obtained from the University of Michigan Committee on Use and Care of Animals according to National Institutes of Health guidelines.
BB/W RAT MODEL.
Prediabetic and nondiabetes-prone male BB/W rats were obtained from the
NIH-sponsored colony at the University of Massachusetts (Worchester,
MA) and maintained at the Michigan Diabetes Research and Training
Center as described previously (Hall et al. 1995b). After the onset of diabetes, ultralente insulin was administered subcutaneously daily to maintain hyperglycemic blood glucose levels between 16 and 25 mM/l (300-450 mg/dl) and prevent ketoacidosis. Blood
glucose levels were monitored daily until the desired plasma glucose
levels were achieved and biweekly thereafter. Age-matched animals
raised in the same facility served as controls. Median nerve conduction
velocity was measured 1-2 wk prior to death, using techniques
described previously (Hall et al. 1995b
).
STZ-INDUCED MODEL.
For studies using streptozotocin-induced diabetic animals, male
Sprague-Dawley rats aged 4-5 mo were rendered diabetic by injection
with 45 mg/kg streptozotocin (Sigma Chemical, St. Louis, MO) as
described previously (Srinivasan et al. 1997).
Age-matched animals injected with vehicle served as controls. Animals
injected with streptozotocin were given 10% sucrose in water for
48 h after injection to prevent hypoglycemia, then maintained on
an ad-lib diet. Blood glucose levels were monitored daily until
induction of diabetes, which usually occurred within 1-3 days, then
biweekly thereafter. Blood glucose ranged from 300 to >550 mg/dl in
STZ-injected animals. Animals were studied between 4 and 8 wk after
induction of diabetes. Weight and blood glucose were measured on the
day of recording.
DRG preparation
Isolated, acutely dissociated thoracic and lumbar DRG neurons
were aseptically prepared using techniques described previously (Hall et al. 1995b) but without the use of nerve growth
factor or horse serum. For electrophysiology experiments, dissociated neurons were plated onto 35-mm sterile culture dishes coated with calf
collagen and incubated in 95% air +5% CO2 at
37°C for 30-60 min prior to performing same-day recordings.
Electrophysiology was completed within 10 h of dissociation. For
GTP
[35S] and opiate receptor binding assays,
purified DRG membranes were prepared from acutely dissociated DRGs
(Szekeres and Traynor 1997
) and tested the same day.
Western immunoblotting for G-protein
subunit expression was
performed on freshly homogenized whole DRG ganglia from paired diabetic
and control animals.
Drug preparation for electrophysiology
All reagents were obtained from Sigma. GTPS and GDP
S were
diluted with ice-cold distilled water to 10 mM. Twenty-microliter aliquots were lyophilized and stored at
20°C. On the recording day,
working solutions of GTP
S (500 µM) and GDP
S (500 µM) were prepared using external recording solution for extracellular
preincubation experiments or at 250 µM with pipette recording medium
for experiments involving intracellular dialysis during the recording.
In experiments using PTX, culture dishes were either preincubated with
medium containing 250 or 500 ng/ml PTX for 3-4 h prior to recording, or recordings were made with PTX (50 ng/ml) added to the recording pipette solution.
[D-Ala2,-Me-Phe4,Gly5-ol]-enkephalin
(DAGO) was prepared as 10-µL aliquots of 10 mM stock solution,
lyophilized, and stored at
70°C. On the recording day, a working
solution of 10 µM was prepared by dilution with external recording
solution containing 0.4% bovine serum albumin fraction V (fatty
acid-free). Solutions were kept on ice until used.
GTP[35S] binding assay
GTP[35S] binding in DRG membranes was
performed using the method of Szekeres and Traynor
(1997)
. All procedures were performed at 0-4°C. DRG
membranes were suspended in buffer A, containing 20 mM HEPES, 100 mM
NaCl, and 4 mM MgCl at pH 7.4, to give a final concentration of 10-15
µg protein/ml (Lowrey et al. 1951
). Membranes were
incubated in Buffer A containing 0.05 nM
GTP
[35S], 50 µM GDP, and varying
concentrations of [D-Ala2, M-Me-Phe4,Gly5]enkephalin-ol; 3-10,000 nM (DAMGO) in a total volume of 100 µl for 30 min at 25°C. Nonspecific binding was defined using unlabeled GTP
S (50 µM). The mixture was rapidly vacuum-filtered through GF/B filters to
separate bound from free ligand. After washing three times in ice-cold
buffer A, radioactivity on the filters was determined by liquid
scintillation counting. Specific binding was typically 90% of total binding.
Opiate receptor binding assay
Mu opioid binding (Szekeres and Traynor 1997) was
assessed in DRG membranes prepared as described in the preceding text.
DRG membranes (10-15 µg/tube) were incubated in Tris-HCl buffer (pH 7.4) with [3H]naloxone at varying
concentrations (1-25 nM) in a final volume of 100 µl for 90 min at
25°C. Naloxone acted as a preferential µ agonist at these
concentrations. Bound and free naloxone were separated by vacuum
filtration through GF/B filters, which were subsequently washed three
times with ice-cold Tris buffer. Nonspecific binding was defined using
unlabeled naloxone (10 µM). Specific binding was typically 80-85%
of total binding at the radioligand Kd.
Binding assay data analysis
Data for both opioid and GTP[35S]
binding were analyzed using the program LIGAND (Munson and
Rodbard 1980
) to provide estimates for the total receptor
number (Bmax), binding affinity
(Kd or Ki), and slope of the binding
isotherms. The EC50 for stimulation of
GTP
[35S] binding obtained at various opioid
concentrations was determined from nonlinear curve fitting of the data
using GraphPAD Prism (GraphPAD, San Diego, CA). Statistical comparisons
between diabetic and control data were performed using the Student's
t-test, where P < 0.05 was considered
significant (Mendenhall 1975
).
Western immunoblot of G-protein subunits
Expression of G-protein subunits was assessed in homogenized
whole thoracic and lumbar dorsal root ganglia obtained from four
control and four BB/W diabetic rats aged 8 ± 2 (SD) mo.
Eight ganglia were obtained from each animal, homogenized in
phosphate-buffered saline (PBS), and prepared according to the method
of Mumby and Gilman (1991)
. Protein concentrations of
homogenized ganglia from each animal were measured (Lowry et al.
1951
) and diluted with buffer to 100 µg/ml. SDS page gel
electrophoresis was performed using 25 µg of tissue per lane. Each
lane consisted of tissue from one animal, and each run included tissue
from a pair of diabetic and age-matched control animals. Molecular
weight markers (Calbiochem, San Diego, CA) and known concentrations of
purified G-protein
subunits (DuPont NEN Research Products, Boston,
MA) were run on the same gel as controls. Immunoblot was performed
using [3H]-labeled rabbit immunoglobulin
directed against the
subunits of Go,
Gi1/2, Gi3, and
Gs (Calbiochem). The pixel densities of the
digitized bands were imaged (Biorad Imaging System, Life Science, Hercules, CA) and analyzed using the Adobe Photoshop program (Adobe Systems, San Jose, CA). Band density measurements were corrected for
background staining by dividing the band density for matched diabetic
and control samples by an equivalent area of background density. To
minimize inter-run variability, pixel density of bands from diabetic
animals were expressed as the percentage difference compared with
age-matched controls run on the same gel using the formula: [1
(diabetic/control)]*100. Similar studies were
performed on DRGs from four STZ-induced rats prepared as described in
the preceding text, and four vehicle-injected controls. Protein loading was 100 µg/lane, and 1 µg of purified Go
protein standard
(Calbiochem) was run as a control.
Whole cell voltage-clamp recordings
Whole cell patch-clamp recordings of high-threshold calcium
currents (ICa) were performed at room
temperature on DRGs between 20 and 40 µm in diameter using techniques
described previously (Hall et al. 1995b). Recording
electrode resistance was 1-2 M
, and seal resistance was 1 G
.
Experiments were performed in a nonperfused culture dish containing the
following external bath solution (in mM): 5 CaCl2, 67 choline Cl, 100 tetraethylammonium chloride, 5.6 glucose, 5.3 KCl, 10 HEPES, and 0.8 MgCl2 (pH 7.3-7.4, 320-330 mosm/kg). In some
experiments, 5 mM BaCl2 replaced
CaCl2. Recording electrodes were filled with (in
mM) 140 cesium Cl, 10 HEPES, 10 EGTA, 5 MgATP, and 0.1 LiGTP (Sigma).
During intracellular dialysis with GDP
S or GTP
S, recording
solutions were prepared with GDP
S or GTP
S added as described in
the preceding text, without additional LiGTP. The pH was adjusted to
7.2-7.3 with 1 M CsOH after addition of ATP and GTP, and the final
osmolality (280-290 mosm/kg) was adjusted to 10-15% below that of
the external recording solution using distilled water. High-threshold
calcium currents were elicited by depolarizing voltage steps generated using the program CLAMPEX (pCLAMP, Axon Instruments, Foster City, CA).
Currents were recorded using an Axopatch 220A patch-clamp amplifier
(Axon Instruments) with an input resistance of 1-3 M
, filtered with
a Bessel filter at 5 kHz (
3 dB), sampled at 20 kHz, and stored on
hard disk as binary data files. The total duration of recording was
10-20 min from the time of patch rupture.
Electrophysiologic stimulation parameters
Neurons were voltage-clamped at a holding potential
(Vh) = 80 mV. Immediately after
patch rupture, whole cell capacitance was measured as described
previously (Hall et al. 1995b
). High-threshold calcium
currents (ICa) were elicited by
depolarization from Vh =
80 to +10
mV for 100 ms. Leak currents were generated by hyperpolarizing commands
of equal value to those used to depolarize the cell. There was no
significant difference in the leak current measured in control (68 ± 44 pA; n = 24) and diabetic neurons (87 ± 32 pA; n = 20). Calcium current density was calculated by
dividing the leak-subtracted ICa by
capacitance to yield (IDCa; pA/pF). This maneuver was performed to control for the effect of diabetic macrosomia on absolute current amplitude (Hall et al.
1995b
). Peak IDCa was
calculated for neurons from diabetic and control animals and compared
using the statistical software package GraphPad Prism (GraphPad
Software). IDCa amplitude and
facilitation of currents were monitored at 30-s intervals by
alternating 100-ms depolarizations to +10 mV with a two-step
facilitation protocol described in the following text. Current-voltage
(I-V) curves and steady-state inactivation curves were
generated using standard techniques (Hall et al. 1995a
).
To determine whether diabetes affected the rate of decay of tail
current deactivation, tail currents from diabetic and control
recordings were normalized by dividing by the maximum tail current
amplitude obtained during each recording. To avoid transient recording
artifacts immediately postdepolarization, maximum tail current
amplitude was measured 50 µs after termination of depolarization.
After normalization, traces were averaged and the best fit for both
diabetic and control recordings was obtained by a Chebyshev fit with
5,000 iterations of a second-order exponential equation of the form
![]() |
Facilitation protocols
Facilitation was elicited using two different protocols designed
to differentiate the effect of calcium-mediated inhibition on
facilitation amplitude. The first (10/90) consisted of a 50-ms prepulse
to +10 mV which was followed in 15 ms by a 25-ms test pulse to +10 mV
(Fig. 4A). After a 3-s delay to allow recovery from
inactivation, a second set of paired pulses was administered, with a
strongly-depolarizing prepulse amplitude of +90 mV, followed by a test
pulse amplitude of +10 mV. Facilitation was defined as the percentage
increase in IDCa amplitude of the
second ("facilitated") test pulse compared with the first test
pulse and was calculated by the following equation
![]() |
![]() |
![]() |
Statistical analysis
Significance was determined by analysis with GraphPad Prism
(GraphPad Software) using two way ANOVA for multiple comparisons and
defined as P < 0.05 (Mendenhall 1975).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of diabetes on nerve conduction velocity and high-threshold calcium currents
Diabetic BB/W rats (diabetic duration, 7-10 mo) had significant
decreases in body weight, elevated glycated hemoglobin (Hb), and
slowing of median nerve conduction velocity (MNCV) compared with
nondiabetic control animals (Table 1).
Animals rendered diabetic by injection of STZ demonstrated similar
changes in weight and blood sugar after 4-5 wk (Table
2). Whole cell, voltage-clamp recordings
of high-threshold calcium current density
(IDCa) were performed on isolated DRG
neurons prepared from BB/W and STZ-induced diabetic animals or
age-matched nondiabetic controls. Currents were elicited at 30-s
intervals using a 100-ms depolarizing voltage step to +10 mV as
described in METHODS. IDCa
increased 20-30% in amplitude during the initial 2-5 min following
patch rupture ("runup") to a maximum amplitude. Maximum
leak-subtracted IDCa amplitude in DRG
neurons from diabetic BB/W and STZ-induced diabetic animals was
significantly enhanced compared with controls (Table 3). Current density subsequently
decreased at a steady rate ("rundown"), such that by 10 min
following patch rupture, the current amplitude was ~60% of the
maximum amplitude recorded. Rundown occurred at a similar rate in
diabetic and control neurons. As we have reported previously
(Hall et al. 1995b), the maximum amplitude of the
current-voltage relationship (I-V) curve was greater in
neurons from BB/W diabetic rats, and there was no shift in the voltage
dependence of activation, steady-state inactivation (data not shown).
There was also no significant difference in the deactivation time
constants (
; ms) of calcium currents recorded in eight control and
eight diabetic BB/W neurons when fit by Boltzman equations with the
following parameters: control:
1 = 0.3 ± 0.1;
2 = 1.9 ± 0.1, diabetic:
1 = 0.4 ± 0.1,
2 = 1.6 ± 0.1. This suggests that there
was no significant difference in the relative proportions of the
various types of whole cell currents activated by depolarization in
diabetic and control DRGs. A similar situation was observed in
recordings from STZ-induced diabetic animals (results not shown).
|
|
|
µ opiate-mediated inhibition of calcium currents was significantly decreased in STZ-induced diabetic DRGs
We had previously observed that opiate-mediated inhibition of
calcium currents in DRGs by dynorphin A was significantly diminished in
neurons from BB/W diabetic rats, compared with age-matched controls
(Hall et al. 1996). Using the µ opiate agonist DAGO, we observed a similar impairment in DAGO-mediated inhibition of calcium
current density in recordings obtained from STZ-induced diabetic DRGs,
compared with age-matched, nondiabetic controls (Fig.
1). Following patch rupture, DRG neurons
were depolarized at 30-s intervals as described in METHODS.
At the time of maximum IDCa amplitude
(usually 2-5 min after patch rupture), 1 µM DAGO was applied by
air-pressure ejection (Hall et al. 1995b
). The effect of
DAGO on calcium current amplitude was assessed within 3 s of
application and at 30-s intervals until the DRG current amplitude had
recovered to pre-DAGO amplitude or until 10 min had elapsed. The point
of maximum inhibition was determined, and the current amplitude
compared with the corresponding pre-DAGO amplitude using a paired
t-test. DAGO application resulted in a significant decrease
in the amplitude of IDCa in DRGs from
control rats (~38%) but not in STZ-induced diabetic animals (15%).
There are several possible explanations for this effect, including
decreased opiate receptor expression, decreased function of the
G-protein-mediated signal pathway coupling the receptor to the calcium
channel, or decreased numbers/function of calcium channels. Our
previous studies suggested that the latter was unlikely as neurons from
both the BB/W and STZ-induced diabetic rat model had enhanced calcium
currents (Hall et al. 1995b
; Ristic et al.
1998
) involving multiple calcium channel subtypes (Hall
et al. 1995b
).
|
DRG µ opiate receptor binding was unchanged in diabetes
We performed µ opiate receptor binding studies using naloxone
(Remmers et al. 1998) to determine whether a decrease in µ receptor expression might be responsible for the diminished opiate
responsiveness in diabetes. The diminished activity was not due to a
decrease in expression of µ opiate receptors, as there was no
significant difference in binding of
[3H]naloxone at µ-selective concentrations to
DRG membrane preparations from STZ-induced diabetic and control animals
(Fig. 2). Because multiple channel
subtypes were affected, we hypothesized that an intermediary pathway,
such as the inhibitory G proteins coupling the receptor to the channel
might be impaired. Subsequent experiments were performed to determine
whether the expression and/or function of coupled G proteins was
altered in diabetes.
|
G-protein expression was not altered in diabetes
Experiments were performed to assess the effect of diabetes on
expression of inhibitory (Gi/o) and stimulatory (Gs) G proteins. Expression of G-protein subunits was assessed in whole ganglia obtained from four control and four BB/W diabetic rats as described in
METHODS. Each lane consisted of tissue from one animal, and each run included tissue from paired diabetic and age-matched control
animals. Representative immunoblots of homogenized whole dorsal root
ganglion neurons from diabetic and nondiabetic BB/W rats incubated with
antibodies directed against Go,
Gi1/2, Gi3 are shown in
Fig. 3A. Total pixel density
of the stained bands corresponding to the G-protein
subunits was
determined for control and diabetic tissue by dividing the pixel
density of the bands by the background staining. Representative pixel
densities for an individual run were Gi1/2:
control, 2,231; diabetic, 2,216, Gi3: control,
1,232; diabetic 1,198, Go: control, 3,610;
diabetic, 3,729, Gs: control, 2,367; diabetic,
2,245. To minimize inter-run variability, total pixel density of
G-protein bands from diabetic DRGs were divided by the pixel density of
the matched control band and expressed as the percentage change from
control. There was no significant effect of diabetes on expression of
these PTX-sensitive G-protein
subunits compared with control DRGs
(Gi1/2:
1.5%; Gi3:
3.5%; Go: +2.9%; Gs:
5%). Similar results were obtained for expression of
Gs
, which was unchanged in diabetes (not shown). There was also no change in expression of Go
observed between DRGs from control and STZ-induced animals (Fig. 3B).
These results indicate that the diminished tonic inhibition of calcium currents observed in neurons from BB/W diabetic animals was unlikely to
be due simply to decreased expression of PTX-sensitive inhibitory G
proteins (Gi/o) or to increased expression of
stimulatory Gs.
|
Voltage-dependent calcium current facilitation was decreased in diabetes
G-protein-mediated inhibition was assessed indirectly using
large-amplitude prepulse facilitation of calcium currents (Ikeda 1991). DRG neurons from BB/W diabetic rats and aged-matched
nondiabetic controls were subjected to facilitation protocols
alternating with 100-ms depolarizing commands performed at 30-s
intervals after patch rupture as outlined in METHODS.
Figure 4 shows the results of
facilitation using a protocol designed to increase calcium-dependent
inhibition (10/90; Fig. 4, A and B) or minimize calcium-dependent inhibition (0/90; Fig. 4, C and
D) of the subsequent test pulse (TP). Using the 10/90-mV
prepulses, percent facilitation of calcium currents was significantly
smaller in DRG neurons from diabetic animals (16 ± 4%,
n = 17, P < 0.05) compared with
controls (28 ± 5%, n = 18; Fig. 4B).
The amplitude of facilitation was relatively stable for the first 5 min
following patch rupture, then decreased with time during the recording,
with a nonsignificant trend toward more rapid decay in control neurons.
Previous studies have demonstrated that facilitation amplitude is
dependent on both the magnitude of the prepulse voltage and the length
of the inter-pulse interval (Ikeda 1991
). We observed
similar voltage- and interval-dependent changes in facilitation
amplitude in our recordings. We observed that the test current
amplitude was often decreased by the preceding +10-mV prepulse when
using the 10/90 protocol (Fig. 4A). We suspected that
calcium entering during the first +10-mV depolarizing prepulse was
causing calcium-dependent inhibition of the +10-mV test pulse and that
the phenomenon of facilitation under these conditions might be
explained by reversal of calcium-dependent inactivation. To test this
hypothesis, we performed two experiments designed to decrease
Ca2+ influx prior to the large-amplitude +90-mV
prepulse. First, a facilitation protocol that did not have a
preliminary +10-mV prepulse was used (Fig. 4, C and
D). Stimulating with facilitation protocols that did not
have a preceding +10-mV prepulse demonstrated that although the
amplitude of facilitation was decreased, neurons from diabetic animals
continued to exhibit significantly smaller percent facilitation of test
currents compared with controls (Fig. 4D). We also performed
experiments on STZ-induced diabetic DRGs and controls in which
Ca2+ in the external recording solution was
replaced by 5 mM Ba2+. Under these conditions,
neurons from diabetic animals continued to exhibit diminished
facilitation compared with controls. This indicated that
calcium-dependent inhibition, while present, could not explain the
difference in facilitation between diabetic and control neurons.
|
Effect of PTX, GTPS, and GDP
S on facilitation
Experiments were performed in BB/W rats to assess whether the
effect of diabetes on facilitation could be altered by opiate ligands
and other pharmacologic agents that modulate G-protein function. The
effects of PTX, GTPS, and GDP
S on facilitation of
IDCa were tested by two methods:
preincubation and intracellular dialysis. Following dissociation and
plating, neurons from control and diabetic animals were preincubated in
culture medium containing PTX (250 ng/ml) for 4 h prior to
recording. As reported previously (Hall et al. 1996
),
PTX preincubation caused a modest, nonsignificant increase in maximum
IDCa in both control and diabetic
neurons. PTX preincubation decreased prepulse facilitation of test
currents by 50% in control DRGs (15 ± 4%) and 35% in diabetic
DRGs (10 ± 4%). Preincubation of both control and diabetic
neurons with GDP
S (500 µM) in the external medium decreased
facilitation by 85%. On the basis of earlier reports (Ikeda
1991
), we had expected that GDP
S would abolish facilitation
completely. Because the effect was incomplete, we speculated that
preincubation may not have allowed adequate uptake of these agents to
demonstrate a maximal effect. Therefore we also performed experiments
in which GDP
S, GTP
S, and PTX were dialyzed into the cells by
adding them to the recording pipette medium. Our previous experience
has shown that such intracellular dialysis reaches a maximum effect in
5-8 min following patch rupture (Hall et al. 1995a
;
Wiley et al. 1997
). Compared to neurons dialyzed with
recording medium containing GTP, intracellular dialysis with GTP
S
(250 µM) caused a rapid rundown in calcium current amplitude by 5 min
(control IDCa: 85 ± 18 pA/pF;
n = 6, diabetic: 100 ± 14 pA/pF;
n = 5, not significantly different). In the presence of
GTP
S (Fig. 5), percent facilitation was increased by a similar amount in control (43 ± 13%) and
diabetic neurons (46 ± 9%), whereas GDP
S dialysis (250 µM)
abolished facilitation in both control and diabetic DRGs. Dialysis with
GTP
S (Fig. 6B) shifted the
voltage range of facilitation to more positive voltages, as indicated
by the increase in the V0.5 (50% of
F/Fmax) listed in the
figure legend, whereas dialysis with 250 µM GDP
S shifted the curve
to more negative voltages. The effect appeared to be analogous to the
rightward voltage shift and slowing of calcium current activation
observed with ligands, such as opiates, that are known to inhibit
calcium currents via a PTX-sensitive G-protein pathway (Bean
1989
). Because larger facilitation amplitudes appear to reflect
increasing preexisting G-protein-mediated inhibition, we anticipated
that the direction of the shift in facilitation for agents such as
GTP
S would be opposite to those described by Bean for whole cell
currents (which decrease in amplitude with increasing
inhibition). These results indicate some degree of reversibility in the
effect of diabetes on G-protein-mediated current facilitation.
|
|
Compared with facilitation in the presence of GTP alone, intracellular dialysis with medium containing GTP and PTX (50 ng/ml; n = 5) decreased facilitation to 16 ± 3% (control) and 11 ± 4% (diabetic; Fig. 5). This decrease was significant in control DRGs but not in DRGs from BB/W diabetic animals. We believe that intracellular dialysis with this concentration of PTX was effective in inhibiting Gi/o-type G proteins, as 1 µM Dyn A or DAGO had no effect on calcium currents in PTX-dialyzed DRGs. Dialysis of PTX concentrations >50 ng/ml appeared to be toxic as reliable recordings could not be performed in neurons exposed to 75 or 100 ng/ml of PTX.
The effect of DAGO treatment on facilitation amplitude provided additional evidence that diabetic inhibitory G-protein pathways appeared to be relatively impaired. We observed that opiate treatment (which presumably activates inhibitory G proteins) was significantly more effective in increasing the amplitude of facilitated currents in control animals (78%), compared with DRGs from STZ-induced diabetic animals (34%; Fig. 7).
|
GTPase activity was diminished in diabetes
Compared with nontreated neurons, DAGO treatment resulted in a
significant increase in percent facilitation in both STZ-induced diabetic (36 ± 5%) and control (74 ± 11%) animals. The
magnitude of the effect of opiate treatment on facilitation of calcium
currents was significantly less in DRGs from diabetic animals compared with controls. We had previously observed that opiate treatment of
diabetic DRGs resulted in significantly less opiate-mediated inhibition
of calcium current amplitude (Hall et al. 1996). These results suggested that the opiate receptor pathway coupled to calcium
channels was significantly impaired in diabetes compared with
nondiabetic controls. Using a direct method to assess GTPase activity
(Traynor and Nahorski 1995
), we observed a significant decrease in the GTPase activity in diabetes as measured by binding of
the µ opiate agonist DAMGO to membranes from diabetic and control rats. The binding affinity of GTP
[35S] in
the presence of DAMGO was significantly (P < 0.01)
shifted to the right in DRGs from diabetic animals (Fig.
8), confirming that opiate stimulation
resulted in diminished GTPase activity in diabetic neurons.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study documents enhanced calcium current amplitude and
diminished facilitation in two models of diabetes, the spontaneous BB/W- and STZ-induced diabetic rat. Voltage-dependent, transient facilitation of calcium currents has been documented in a variety of
neuronal and nonneuronal cells (Dolphin 1996;
Ikeda 1991
). Facilitation may have physiologic
significance in neuronal circuits in which simultaneous depolarizing
inputs may transiently elevate membrane potential to large positive
values (Cens et al. 1998
). The mechanism is thought to
involve transient voltage-dependent dissociation of inhibitory
G-protein
subunits from the proposed effector site on the
calcium channel (I-II intra-cytoplasmic loop) (De Waard et al.
1997
; Garcia et al. 1998
). The amplitude of
facilitation decays rapidly with increasing inter-pulse intervals. This
suggests that facilitation represents a tightly coupled,
membrane-delimited phenomenon (De Waard et al. 1997
)
comparable to that observed in kinetic studies of opiate
receptor-G-protein coupling (Wilding et al. 1995
).
Diabetes was associated with a significant decrease in
voltage-dependent facilitation of calcium currents compared with
age-matched control neurons. Facilitation was decreased by
intracellular dialysis with PTX and abolished by dialysis with GDPS.
The elevated calcium currents and the diminished facilitation observed
in diabetes were restored to control levels by intracellular dialysis
with GTP
S, suggesting that the impairment in G-protein-mediated
inhibition observed in diabetes was reversible. The voltage dependence
of facilitation was shifted to the right in diabetes compared with controls, suggesting a relative impairment in elicitation of the response. This effect may be analogous to voltage shifts in calcium currents observed in the presence of inhibitory ligands that have been
interpreted as G-protein-mediated shifts of the calcium channel to a
more "reluctant" state (Bean 1989
). Modulation of
G-protein activity by dialysis with GTP
S and GDP
S resulted in
shifts in voltage dependence of facilitation that appeared to
correspond with more (left shift) or less (right shift) inhibition,
respectively. In the presence of GTPase modulators,
IDCa and facilitation amplitudes were
similar in diabetic and control neurons. Expression of PTX-sensitive G-protein
subunits (Gi1/2,
Gi3, and Go) was not
affected by diabetes, suggesting that impaired function of coupled G
proteins, rather than decreased expression, underlies the increased
calcium currents in diabetes (Hall et al. 1995b
). This
was confirmed using a direct assay of GTPase activity, which
demonstrated a significant impairment in opiate-mediated
GTP
[35S] uptake, but not µ receptor
expression, in diabetic DRGs. We believe the present study provides
functional evidence of impairment in G-protein-coupled calcium current
modulation in two models of diabetic neuropathy and indicates a
potential mechanism for the impairment in calcium current facilitation
observed when nondiabetic DRGs are exposed to diabetic serum
(Ristic et al. 1998
).
The facilitation response evoked by large amplitude depolarizations in
rat DRGs appears, in part, to involve reversal of calcium-dependent inactivation of calcium currents. Under conditions promoting
Ca2+-dependent inactivation (Fig. 4A),
the test current was often smaller than the preceding prepulse current
despite the identical amplitude of the voltage steps. This diminution
was not observed if test currents were elicited under conditions
designed to minimize Ca2+-dependent inactivation:
i.e., the 0/90 facilitation protocol (Fig. 4C) or recording
in Ba2+-containing solutions. Because increased
calcium influx would be expected to result in increased
inhibition by this mechanism (i.e., increased facilitation),
calcium-dependent inhibition of currents does not explain the
observation that facilitation is decreased in diabetes. Facilitation in
skeletal muscle from streptozotocin-induced diabetic rats has been
attributed to increased numbers of calcium channels (Ogawa et
al. 1995). The almost-complete reversal of the effects of
diabetes on calcium currents in the presence of analogues that modulate
G-protein activity argues against an absolute increase in the number of
calcium channels in diabetes. Decreased opiate receptor expression
could also account for impaired opiate-mediated inhibition of calcium
channels in diabetes (Hall et al. 1996
). However, our
preliminary data in the STZ-induced diabetic model indicate that µ opiate receptor expression on DRGs is increased, not decreased, in
diabetes (unpublished observations). Expression studies in
Xenopus oocytes demonstrate that voltage-dependent facilitation is independent of inhibitory receptor expression and
function (De Waard et al. 1997
).
Our results suggest that both PTX-sensitive and -insensitive G proteins
may be involved in calcium current inhibition. Facilitation was
decreased by dialysis with PTX and abolished by dialysis with GDPS.
Based on previous reports, Go is a likely
candidate for the PTX-sensitive component (Moises et al.
1994
; Wiley et al. 1997
). The PTX-insensitive
G-protein species mediating tonic inhibition of neuronal calcium
channels remains to be characterized. The mechanism of facilitation has
been proposed to involve a voltage-dependent, transient uncoupling of
the G-protein
subunit complex from a binding site on the calcium
channel (Reuveny et al. 1994
; Stanley and
Mirotznik 1997
). The binding site may be close to, or identical with, the location of calcium channel
subunit-binding site on the
I-II intracytoplasmic loop of the
1 calcium channel subunit (Cens et al. 1998
). Alterations in the binding
characteristics of the subunits involved, whether on the calcium
channel or G protein, could explain the changes in calcium current
amplitude and facilitation. Interest has centered on phosphorylation,
particularly by protein kinase C (PKC), as a potential mechanism for
modulation of G-protein-calcium channel function. Calcium currents are
increased by treatment with PKC (Hall et al. 1995a
;
Sculptoreanu et al. 1995
), although it is not clear
whether such effects are due to phosphorylation of consensus sites on
the channel or on the G protein. A recent report indicates that PKC can
modulate calcium channels by phosphorylation of two separate sites on
the I-II domain of the channel
subunit (Hamid et al.
1999
). Mutation of one site abolished the ability of PKC to
increase calcium currents but had no effect on G-protein-mediated
inhibition, whereas mutation of a separate site on the channel
prevented G-protein-mediated inhibition of calcium currents. Our
results indicate that increased calcium current density in diabetic
neuropathy is associated with a relative decrease in the ability of G
proteins to inhibit channels. This effect appears to be reversible.
G-protein-mediated modulation of channels could be decreased in
diabetes by PKC-mediated phosphorylation of the G-protein-associated
site on the calcium channel (Hamid et al. 1999
) or the G
proteins themselves (Katada et al. 1985
). Increased
phosphorylation of the PTX-sensitive Gi2
subunit has been reported in hepatocytes of STZ-induced diabetic rats (Morris et al. 1996
); however, it remains to be seen
whether the Go-subtype coupled to opiate
receptors in DRG neurons is similarly affected.
We favor the interpretation that the enhancement in calcium influx in
diabetes (Hall et al. 1995b,1996
; Levy et al.
1994
) is pathophysiologic rather than compensatory. The calcium
"set point" hypothesis (Orrenius and Nicotera 1994
)
suggests that modest changes in cytosolic calcium
([Ca2+]i) over prolonged
periods may be injurious. Our previous studies demonstrated that the
magnitude of enhancement increased with diabetic duration and was
correlated with increasing impairment in nerve conduction velocity
(Hall et al. 1995b
). Treatment of new onset BB/W
diabetic animals with long-term administration of an aldose reductase
inhibitor was associated with normalization of nerve conduction
velocity and neuronal calcium influx despite persistent hyperglycemia.
Exposure to diabetic serum is associated with elevated calcium influx
and increased programmed, apoptotic cell death (Prittinger et
al. 1998
; Srinivasan et al. 1998
), both of which
can be prevented by short-term treatment with calcium channel
antagonists (Srinivasan et al. 1998
). In addition,
treatment with diabetic serum increased calcium current density and
decreased facilitation in nondiabetic neurons in a manner similar to
that reported in this study (Ristic et al. 1998
). This
suggests that autoimmune factors in serum may be the underlying cause
of abnormal calcium signaling in diabetes.
In summary, the results of this study suggest that the function of the
inhibitory G-protein-calcium channel complex is impaired in diabetes,
providing one explanation for the abnormal calcium signaling described
previously (Biessels and Gispen 1996; Hall et al.
1995b
, 1996
; Levy et al. 1994
). Altered
G-protein function in diabetes was not due to a relative decrease in
expression of PTX-sensitive inhibitory G proteins
(Gi/o), and the functional changes were
reversible, as maximal (or near-maximal) activation of G proteins by
GTP
S resulted in equivalent enhancement of inhibitory G-protein
function in neurons from both control and diabetic animals. This study
indicates that a significant impairment in GTPase function is the
likely explanation for the decreased efficacy of opiate-mediated inhibition in diabetes.
![]() |
ACKNOWLEDGMENTS |
---|
We thank B. Dzwonek, H. Sheng, A. Merry, and H. Ristic for expert technical assistance.
These studies were supported by a Michigan Peptide Research Center Pilot Feasibility Award and Juvenile Diabetes International Research Award 195036 to K. E. Hall, a Veterans Affairs Merit Award, University of Michigan Diabetes Research and Training Center Pilot Feasibility Award to J. W. Wiley, and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-45820-01 (J. W. Wiley) and DK-43884 (A.A.F. Sima).
![]() |
FOOTNOTES |
---|
Address for reprint requests: K. E. Hall, VA Medical Center, GRECC 11G, D-318, Ann Arbor, MI 48105 (E-mail: kehall{at}umich.edu).
Received 6 November 2000; accepted in final form 2 May 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|