Impaired Inhibitory G-Protein Function Contributes to Increased Calcium Currents in Rats With Diabetic Neuropathy

Karen E. Hall,1,2 Jackie Liu,2 Anders A. F. Sima,3 and John W. Wiley1,2

 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


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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 GTPgamma S 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 GTPgamma S, decreased by pertussis toxin, and abolished by GDPbeta S within 5 min. Direct measurement of GTPase activity using opiate-mediated GTPgamma [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 alpha  subunits. These studies indicate that impaired regulation of calcium channels by G proteins is an important mechanism contributing to enhanced calcium influx in diabetes.


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INTRODUCTION
METHODS
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DISCUSSION
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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.


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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 GTPgamma [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 alpha  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. GTPgamma S and GDPbeta 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 GTPgamma S (500 µM) and GDPbeta 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.

GTPgamma [35S] binding assay

GTPgamma [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 GTPgamma [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 GTPgamma 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 GTPgamma [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 GTPgamma [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 alpha  subunits

Expression of G-protein alpha  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 alpha  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 alpha  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 Goalpha 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 MOmega , and seal resistance was 1 GOmega . 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 GDPbeta S or GTPgamma S, recording solutions were prepared with GDPbeta S or GTPgamma 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 MOmega , 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
<IT>Y</IT><IT>=</IT><IT>A</IT><IT> ∗ exp</IT>((<IT>k</IT><SUB><IT>1</IT></SUB><IT>−</IT><IT>t</IT>)<IT>/&tgr;<SUB>1</SUB></IT>)<IT>+</IT><IT>B</IT><IT> ∗ exp</IT>((<IT>k</IT><SUB><IT>2</IT></SUB><IT>−</IT><IT>t</IT>)<IT>/&tgr;<SUB>2</SUB></IT>)<IT>+</IT><IT>C</IT>
where tau 1 and tau 2 were the time constants of the fast and slow decay components in milliseconds. Parameters for the control curve (n = 8) were: A = 1.3915; k1 = 0.00048; B = 1.0687; k2 = 0.05263; C = -0.97646, diabetic curve (n = 8): A = 0.68774; k1 = 0.001733; B = 0.68064; k2 = 0.03943; C = -0.16446.

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
((second test pulse−first test pulse)/first test pulse) ∗ 100
A second protocol (0/90), consisting of a "null" prepulse (0 depolarization/+10 mV test pulse) followed in 3 s by a +90 mV/+10 mV pair, was employed to minimize the amount of calcium entering prior to facilitation (Fig. 4C). The voltage range of facilitation was determined by sequentially increasing the large-amplitude prepulse depolarization voltage from -20 to +90 mV and plotting percent facilitation of the test pulse against prepulse voltage amplitude. Normalized activation curves for percent facilitation in control and diabetic neurons were generated by dividing facilitation amplitude by maximum facilitation elicited by a +90-mV prepulse. Curves were fit using a Boltzman equation of the form
<IT>F</IT><IT>/</IT><IT>F</IT><SUB><IT>max</IT></SUB><IT>=1/</IT>(<IT>1+exp</IT>((<IT>V</IT><SUB><IT>50</IT></SUB><IT>−</IT><IT>V</IT>)<IT>/</IT><IT>k</IT>))
where F = facilitation amplitude at prepulse voltage V, Fmax = maximum facilitation amplitude, V50 = prepulse voltage at 1/2 Fmax, V = prepulse voltage, and k = slope. The effect of varying the inter-pulse interval on facilitation amplitude was determined by sequentially increasing the inter-pulse interval from 12 to 500 ms, while keeping the prepulse amplitude constant at +90 mV. Normalized facilitation amplitude (facilitation/maximum facilitation obtained with a +90-mV prepulse; 12-ms inter-pulse interval) was plotted versus increasing inter-pulse interval. The best fit of the data was obtained with a double exponential equation of the form
<IT>F</IT><IT>=</IT><IT>A</IT><IT> ∗ exp</IT>(−<IT>k</IT><SUB><IT>1</IT></SUB><IT> ∗ </IT><IT>T</IT>)<IT>+</IT><IT>B</IT><IT> ∗ exp</IT>(−<IT>k</IT><SUB><IT>2</IT></SUB><IT> ∗ </IT><IT>T</IT>)
where F = normalized facilitation amplitude at prepulse interval T, A and B = time constants, and k1 and k2 = decay rate constants.

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).


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ABSTRACT
INTRODUCTION
METHODS
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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 (tau ; ms) of calcium currents recorded in eight control and eight diabetic BB/W neurons when fit by Boltzman equations with the following parameters: control: tau 1 = 0.3 ± 0.1; tau 2 = 1.9 ± 0.1, diabetic: tau 1 = 0.4 ± 0.1, tau 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).


                              
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Table 1. Body weight and nerve conduction velocity significantly decreased in diabetic BB/W rats


                              
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Table 2. Body weight and blood glucose in STZ-induced diabetic rats compared to controls


                              
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Table 3. Enhanced calcium current density (IDCa) in DRGs from BB/W and STZ-induced diabetic rats

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).



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Fig. 1. Effect of 1 µm [D-Ala2,-Me-Phe4,Gly5-ol]-enkephalin (DAGO) on calcium current amplitude in control and streptozotocin (STZ)-induced diabetic dorsal root ganglion (DRG) neurons. Left: the means ± SE of 10 recordings performed as described in METHODS in control and diabetic DRGs prior and subsequent to application of DAGO. Representative tracings from a control DRG and diabetic DRG are displayed on the right. Prior to DAGO application, mean peak calcium current density was significantly greater in DRGs from diabetic animals (* P < 0.05) compared with controls. Compared to pre-DAGO currents, control DRGs demonstrated a significant decrease in peak calcium current density amplitude (*** P < 0.01). Neurons from diabetic animals had a small, nonsignificant decrease in current density post-DAGO, compared with pre-DAGO levels.

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.



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Fig. 2. Mu receptor binding was similar in neurons from STZ-induced diabetic and control DRGs as measured by equilibrium binding of [3H] naloxone. Each experiment consisted of paired binding studies performed as described in METHODS in 1 diabetic and 1 control animal. Results are the averages ± SE for 4 separate experiments.

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 alpha  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 alpha  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 alpha  subunits compared with control DRGs (Gi1/2: -1.5%; Gi3: -3.5%; Go: +2.9%; Gs: -5%). Similar results were obtained for expression of Gsalpha , which was unchanged in diabetes (not shown). There was also no change in expression of Goalpha 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.



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Fig. 3. Expression of the alpha  subunits of Gi1/2, Gi3, and Go was not affected by diabetes. A: representative Western blots of BioBred/Worchester (BB/W) rat DRG homogenates stained with radiolabeled antibodies directed against the alpha  subunits of Gi1/2, Gi3, and Go. Left: molecular weight. Each band contains 25 µg of homogenate. Bands corresponding to the appropriate G-protein alpha  subunit (left-arrow ) were digitally scanned and pixel density measured and corrected for background staining. There was no significant difference in the band density of paired samples from BB/W and aged matched control rats. B: Western blot of Goalpha expression in paired samples from a STZ and control sham-injected animal (protein loading, 100 µg homogenate/lane) did not demonstrate any difference in expression. Bands were observed at the same location as 1 µg of purified Goalpha protein standard (S).

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.



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Fig. 4. Amplitude and time course of facilitation in control and BB/W diabetic DRGs. Left: facilitation protocols and representative current tracings; right: percent facilitation vs. time from patch rupture. A: +10/90-mV prepulse depolarization protocol. Two superimposed current tracings are displayed below the corresponding depolarization protocols; one corresponding to a prepulse (PP) voltage of +10 mV followed by a nonfacilitated +10-mV test pulse (TP) and a second with a prepulse voltage of +90 mV and subsequent larger facilitated +10-mV test pulse (). B: percent facilitation of the TP current was determined at 1-min intervals for control (open circle ) and diabetic () DRGs and plotted vs. time from patch rupture. Facilitation decreased with time from patch rupture. Insets: the means ± SE measured at the peak of percent facilitation, which was significantly decreased in diabetic neurons (DM; n = 17; * P < 0.05) compared with controls (C; n = 18). C: 0/90-mV prepulse depolarization protocol with two superimposed current tracings; one corresponding to a prepulse voltage of 0 mV followed by a nonfacilitated +10 mV test pulse, and a second with a prepulse voltage of +90 mV and subsequent larger facilitated +10-mV test pulse (). D: percent facilitation of TP was diminished when protocols without an initial +10-mV prepulse were used. Inset: mean + SE of peak percent facilitation (n = 17) in the first 5 min following patch rupture (inset) was significantly (* P < 0.05) less in diabetic DRGs compared with controls using the 0/90 facilitation protocol.

Effect of PTX, GTPgamma S, and GDPbeta 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, GTPgamma S, and GDPbeta 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 GDPbeta S (500 µM) in the external medium decreased facilitation by 85%. On the basis of earlier reports (Ikeda 1991), we had expected that GDPbeta 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 GDPbeta S, GTPgamma 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 GTPgamma 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 GTPgamma S (Fig. 5), percent facilitation was increased by a similar amount in control (43 ± 13%) and diabetic neurons (46 ± 9%), whereas GDPbeta S dialysis (250 µM) abolished facilitation in both control and diabetic DRGs. Dialysis with GTPgamma 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 GDPbeta 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 GTPgamma 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.



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Fig. 5. Effect of intracellular dialysis with GTPgamma S, GDPbeta S, and PTX on facilitated calcium current density. The graph shows the means ± SE facilitation recorded in neurons from control and BB/W diabetic animals after 5 min intracellular dialysis with GTP, GTPgamma S, GTP + PTX, and GDPbeta S. With GTP dialysis, percent facilitation of the test pulse current amplitude was significantly smaller (* P < 0.05) in DRGs from diabetic rats compared with controls. Percent facilitation of currents in analogue or PTX-treated neurons were compared against the corresponding GTP-treated group for statistical significance (i.e., diabetic GTPgamma S vs. diabetic GTP). Dialysis with GTPgamma S significantly increased percent facilitation in both control (n = 6) and diabetic (n = 5) neurons compared with the corresponding GTP-dialyzed neurons (control; n = 18; diabetic; n = 17). Compared to GTP-dialyzed neurons, PTX dialysis decreased facilitation (control, n = 5; diabetic, n = 5). The decrease was significant in control DRGs but not in diabetic DRGs. Dialysis with GDPbeta S abolished facilitation in both control (n = 4) and diabetic (n = 5) neurons (* P < 0.05, ** P < 0.01, *** P < 0.005).



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Fig. 6. Voltage dependence of facilitation in BB/W diabetic and nondiabetic control DRGs. A: representative tracing of the voltage-dependent facilitation protocol with the depolarizing voltages shown above the corresponding facilitated calcium current tracing. Prepulse voltages were increased from -20 to +90 mV, resulting in a step-wise fashion (facilitation) in the test pulse current amplitude. For clarity, only current tracings from 0 to +90 are shown. B: the voltage dependence of facilitation was calculated for test pulse amplitudes normalized to maximum amplitude (F/Fmax) and plotted as a function of the prepulse voltage for 11 control (C) and 11 diabetic (D) neurons dialyzed with 100 µM GTP, 6 control and 6 diabetic DRGs dialyzed with 250 µM GTPgamma S, and 4 control and 4 diabetic DRGs dialyzed with 250 µM GDPbeta S. At 50% F/Fmax, diabetes was associated with a significant (P < 0.05) positive shift (V0.5 = 22.2 ± 3.5) in the voltage dependence of facilitation (mV) compared with control recordings (V0.5 = 13.7 ± 3.4). Intracellular dialysis with GTPgamma S produced a rightward shift (C + GTPgamma S = 35.2 ± 2.4; D + GTPgamma S = 40.9 ± 4.1), while GDPbeta S dialysis (C + GDPbeta S = 8.0 ± 3.1; D + GDPbeta S = 7.9 ± 3.5) was associated with a leftward shift in the voltage dependence of 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).



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Fig. 7. Compared to untreated neurons, DRGs from control (n = 5) and STZ-induced diabetic (n = 5) animals treated with 1 µM DAGO demonstrated significantly increased percent facilitation of test pulse currents within 1 min of application. Representative recordings of the effect of DAGO on test currents in a control and STZ-induced diabetic DRG are shown on the right. * P < 0.05; ** P < 0.01; *** P < 0.001.

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 GTPgamma [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.



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Fig. 8. GTPase activity was measured using binding of GTPgamma [35S] in the presence of DAMGO as described in METHODS. Neurons from STZ-induced diabetic animals exhibited significantly lower DAMGO-stimulated binding compared with nondiabetic controls with a significant (P < 0.01) leftward shift in the binding affinity.


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INTRODUCTION
METHODS
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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 beta gamma 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 GDPbeta S. The elevated calcium currents and the diminished facilitation observed in diabetes were restored to control levels by intracellular dialysis with GTPgamma 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 GTPgamma S and GDPbeta 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 alpha  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 GTPgamma [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 GDPbeta S. 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 beta gamma 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 beta  subunit-binding site on the I-II intracytoplasmic loop of the alpha 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 alpha  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 Gi2alpha 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 GTPgamma 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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society