Stress and glucocorticoid receptor transcriptional programming in time and space: Implications for the brain-gut axis.

BACKGROUND: Chronic psychological stress is associated with enhanced abdominal pain and altered intestinal barrier function that may result from a perturbation in the hypothalamic-pituitary-adrenal (HPA) axis. The glucocorticoid receptor (GR) exploits diverse mechanisms to activate or suppress congeneric gene expression, with regulatory variation associated with stress-related disorders in psychiatry and gastroenterology. PURPOSE: During acute and chronic stress, corticotropin-releasing hormone drives secretion of adrenocorticotropic hormone from the pituitary, ultimately leading to the release of cortisol (human) and corticosterone (rodent) from the adrenal glands. Cortisol binds with the GR in the cytosol, translocates to the nucleus, and activates the NR3C1 (nuclear receptor subfamily 3, group C, member 1 [GR]) gene. This review focuses on the rapidly developing observations that cortisol is responsible for driving circadian and ultradian bursts of transcriptional activity in the CLOCK (clock circadian regulator) and PER (period circadian clock 1) gene families, and this rhythm is disrupted in major depressive disorder, bipolar disorder, and stress-related gastrointestinal and immune disorders. Glucocorticoid receptor regulates different sets of transcripts in a tissue-specific manner, through pulsatile waves of gene expression that includes occupancy of glucocorticoid response elements located within constitutively open spatial domains in chromatin. Emerging evidence supports a potentially pivotal role for epigenetic regulation of how GR interacts with other chromatin regulators to control the expression of its target genes. Dysregulation of the central and peripheral GR regulome has potentially significant consequences for stress-related disorders affecting the brain-gut axis.

Corticosterone mediates stress-related increased intestinal permeability in a region-specific manner.

BACKGROUND: Chronic psychological stress (CPS) is associated with increased intestinal epithelial permeability and visceral hyperalgesia. It is unknown whether corticosterone (CORT) plays a role in mediating alterations of epithelial permeability in response to CPS. METHODS: Male rats were subjected to 1-h water avoidance (WA) stress or subcutaneous CORT injection daily for 10 consecutive days in the presence or absence of corticoid receptor antagonist RU-486. The visceromotor response (VMR) to colorectal distension (CRD) was measured. The in situ single-pass intestinal perfusion was used to measure intestinal permeability in jejunum and colon simultaneously. KEY RESULTS: We observed significant decreases in the levels of glucocorticoid receptor (GR) and tight junction proteins in the colon, but not the jejunum in stressed rats. These changes were largely reproduced by serial CORT injections in control rats and were significantly reversed by RU-486. Stressed and CORT-injected rats demonstrated a threefold increase in permeability for PEG-400 (MW) in colon, but not jejunum and significant increase in VMR to CRD, which was significantly reversed by RU-486. In addition, no differences in permeability to PEG-4000 and PEG-35 000 were detected between control and WA groups. CONCLUSIONS & INFERENCES: Our findings indicate that CPS was associated with region-specific decrease in epithelial tight junction protein levels in the colon, increased colon epithelial permeability to low molecular weight macromolecules which were largely reproduced by CORT treatment in control rats and prevented by RU-486. These observations implicate a novel, region-specific role for CORT as a mediator of CPS-induced increased permeability to macromolecules across the colon epithelium.

Reciprocal changes in vanilloid (TRPV1) and endocannabinoid (CB1) receptors contribute to visceral hyperalgesia in the water avoidance stressed rat.

BACKGROUND: Increasing evidence suggests that chronic stress plays an important role in the pathophysiology of several functional gastrointestinal disorders. We investigated whether cannabinoid receptor 1 (CB1) and vanilloid receptor 1 (TRPV1; transient receptor potential vanilloid 1) are involved in stress-induced visceral hyperalgesia. METHODS: Male rats were exposed to 1 h water avoidance (WA) stress daily for 10 consecutive days. The visceromotor response (VMR) to colorectal distension (CRD) was measured. Immunofluorescence and western blot analysis were used to assess the expression of CB1 and TRPV1 receptors in dorsal root ganglion (DRG) neurons. RESULTS: WA stressed rats demonstrated a significant increase in the serum corticosterone levels and faecal pellet output compared to controls supporting stimulation of the hypothalamic-pituitary-adrenal (HPA) axis. The VMR increased significantly at pressures of 40 and 60 mm Hg in WA stress rats compared with controls, respectively, and was associated with hyperalgesia. The endogenous CB1 agonist anandamide was increased significantly in DRGs from stressed rats. Immunofluorescence and western blot analysis showed a significant decrease in CB1 and a reciprocal increase in TRPV1 expression and phosphorylation in DRG neurons from stressed rats. These reciprocal changes in CB1 and TRPV1 were reproduced by treatment of control DRGs with anandamide in vitro. In contrast, treatment of control DRGs in vitro with the CB1 receptor agonist WIN 55,212-2 decreased the levels of TRPV1 and TRPV1 phosphorylation. Treatment of WA stress rats in situ with WIN 55,212-2 or the TRPV1 antagonist capsazepine prevented the development of visceral hyperalgesia and blocked the upregulation of TRPV1. CONCLUSIONS: These results suggest that the endocannabinoid (CB1) and TRP (TRPV1) pathways may play a potentially important role in stress-induced visceral hyperalgesia.

The many faces of nitric oxide: cytotoxic, cytoprotective or both.

Nitric oxide (NO) has emerged as a major modulator of cellular function in health and disease. In addition to its well-known role as a mediator of smooth muscle relaxation, a rapidly developing body of research suggests, paradoxically, that NO can have both cytotoxic and cytoprotective effects. In this issue of Neurogastroenterology and Motility, Choi et al. provide evidence that supports NO has a prosurvival effect on interstitial cells of Cajal in the mouse stomach. The objective of this short review is to place this interesting report in the context of the current literature.

Diabetic autonomic neuropathy: evidence for apoptosis in situ in the rat.

We examined the hypothesis that activation of the apoptosis cascade occurs relatively early in diabetes mellitus affecting three distinct neuronal populations that are involved in regulating gut function: (i) dorsal root ganglion (DRG), (ii) vagus nodose ganglion and (iii) colon myenteric plexus. A validated streptozotocin-induced diabetic rat model and age-matched healthy controls were studied. After 4-8 weeks of diabetes the animals were anaesthetized, fixed in situ and the relevant tissues removed. After 1 month of diabetes some animals were treated with insulin for 2 weeks to restore euglycaemia. Apoptosis was measured using immunohistochemical detection of activated caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL)-positive cells in adjacent sections in neurones (PGP 9.5-positive cells). The level of apoptosis was confirmed using double-label assessment of caspase-3 and TUNEL in DRG preparations. Caspase-3 immunoreactive neurones demonstrated a range in staining intensity. When all grades of staining were included, 6-8% of the DRG, nodose ganglia and myenteric neurones were immunoreactive in the preparations from diabetic rats compared with 0.2-0.5% in controls. Neurones staining positive for both caspase-3 and TUNEL accounted for 1-2% of the total neuronal population in all three preparations in diabetic rats compared with 0.1-0.2% in controls (P < 0.05). Insulin treatment reversed the percentage of TUNEL-positive neurones in diabetic rats to control levels. Activation of the apoptosis cascade occurs relatively early in diabetic autonomic neuropathy and may contribute to the pathophysiology of this disorder.

Impaired inhibitory G-protein function contributes to increased calcium currents in rats with diabetic neuropathy.

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 mu opiate receptors via inhibitory G(o)-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 GTPgammaS 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 (I(DCa)) 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 GTPgammaS, decreased by pertussis toxin, and abolished by GDPbetaS within 5 min. Direct measurement of GTPase activity using opiate-mediated GTPgamma[(35)S] 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 mu 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.

Treatment of aged rat sensory neurons in short-term, serum-free culture with nerve growth factor reverses the effect of aging on neurite outgrowth, calcium currents, and neuronal survival.

Impaired NGF production and release has been documented in aged animals, suggesting that decreased NGF receptor stimulation may be one factor contributing to neuronal dysfunction with aging. Other studies have suggested that aging may be associated with impaired intracellular responses to NGF. Because aging-associated neuronal dysfunction contributes to morbidity and mortality in the geriatric population, it is important to determine whether the effects of aging on sensory neuron function and survival are reversible. In the present study, we observed significantly decreased neurite outgrowth and neuronal survival in short-term cultures (0-96 h) of dorsal root ganglion (DRG) neurons from aged (>22 months) Fisher 344 x Brown Norway F1 hybrid rats, compared to young (4-6 month) and middle-aged (14 month) animals. From 24 to 96 h in culture, diminished survival of aged neurons appeared to be due to an increased rate of apoptotic cell death. DRG neurons from aged animals also exhibited significantly decreased whole cell, high-threshold voltage-dependent calcium currents, with a larger proportion of L-type current, compared to youthful and middle-aged animals. Treatment of aged DRG neurons with NGF restored neurite outgrowth, neuronal survival and calcium current amplitude and subtype distribution to those observed in youthful DRG neurons.

Decreased expression of nitric oxide synthase in the colonic myenteric plexus of aged rats.

Nitric oxide (NO) is a major non-adrenergic, non-cholinergic (NANC) inhibitory neurotransmitter in the gastrointestinal tract. NO released from the myenteric plexus enhances colonic transit and facilitates propulsion of the colonic contents by mediating descending relaxation. Although it has been suggested that colonic transit delays with aging, the mechanism of delayed colonic transit in aging remains unclear. We hypothesized that advanced age is associated with decreased expression of neuronal NO synthase (nNOS) and concomitant reduction in synthesis of NO in the rat colon. We studied nNOS mRNA expression, nNOS-immunohistochemistry, nNOS-immunoblotting and NOS catalytic activity in the mid-colon obtained from young (age 4-8 months) and aged (age 22-28 months) Fisher (F344xBN)F1 rats. Western blot analysis of PGP 9.5, a generic neuronal marker, of the colonic tissues were employed to study whether the total number of neurons of the myenteric plexus is reduced with aging. The number of nNOS-immunoreactive cells and nNOS synthesis in the colonic myenteric plexus were significantly reduced in aged rats. In contrast, expression of PGP 9.5 in colonic tissues was not affected in aged rats. Northern blot analysis demonstrated that the expression of neuronal nNOS mRNA was significantly reduced in the colonic tissues in aged rats. Basal and veratridine-induced release of L-[(3)H]citrulline were significantly decreased in colonic tissues from aged rats, compared to young rats. It is suggested that advanced age is associated with diminished gene expression of nNOS, nNOS synthesis and catalytic activity of NOS. This may explain the mechanism of delayed colonic transit observed in advanced age.

Diabetic peripheral neuropathy: evidence for apoptosis and associated mitochondrial dysfunction.

We hypothesized that diabetic sensory neuropathy is associated with activation of apoptosis and concomitant mitochondrial dysfunction. Studies were performed in excised intact and acutely dissociated dorsal root ganglion (DRG) neurons from control and streptozotocin-induced diabetic rats with decreased peripheral nerve conduction velocities (NCV). Apoptosis was increased in acutely dissociated DRG neurons from 3- to 6-week-old diabetic rats. Basal mitochondrial membrane potential (deltapsi) was significantly more positive in DRG neurons from diabetic rats. Depolarization with glutamate resulted in significantly more positive deltapsi and delayed recovery of deltapsi in neurons from diabetic rats. Restoration of euglycemia for 2 weeks with insulin implants normalized NCV, deltapsi, and apoptosis. Intact and acutely dissociated neurons from diabetic rats demonstrated decreased Bcl-2 levels and translocation of cytochrome C from the mitochondria to the cytoplasm. Neither levels of Bax nor levels of Bcl-XL were altered in diabetic neuropathy. Apoptosis associated with mitochondrial dysfunction may contribute to the pathogenesis of diabetic sensory neuropathy.

Effectiveness of the boston brace in treatment of large curves in adolescent idiopathic scoliosis.

STUDY DESIGN: This is a retrospective study of 50 patients with adolescent idiopathic scoliosis with curves measuring 35 degrees to 45 degrees who were treated with a Boston brace. OBJECTIVES: The purpose of this study was to determine whether the Boston brace could effectively halt long-term progression in skeletally immature adolescents with idiopathic scoliosis who had a curve between 35 degrees and 45 degrees. SUMMARY OF BACKGROUND DATA: The Boston brace has been shown to be effective in preventing curve progression in adolescent idiopathic scoliosis, but its efficacy in large curves has not been fully studied. METHODS: Fifty adolescents were treated with a Boston brace for idiopathic scoliosis curves of 35-45 degrees (mean, 38.55 degrees ). All were judged to be skeletally immature based on menarcheal status (mean, 2.6 months before menarche), Risser sign (mean, 0.90; range, 0-2), and chronologic age (mean, 13 +/- 1 years). Patients were recalled for long-term follow-up at a mean of 9.7 years (range, 6.23-13.22 years) after brace discontinuation. Three well-matched patient subsets were then identified based on compliance. Group 1 (n = 24) consisted of patients who were compliant with the brace program and wore the brace 18 or more hours per day, Group 2 (n = 14) contained patients who wore the brace 12-18 hours per day, and Group 3 (n = 12) contained patients who wore the brace 0-12 hours per day. RESULTS: There was a significant difference in the amount of initial correction seen in the brace between the groups: 49%, 45%, and 33% curve correction in the brace for Groups 1, 2, and 3, respectively (P < 0.05). At long-term follow-up there was a statistically significant difference between Groups 1, 2, and 3 in the percentage of patients in whom the curve had progressed to more than 45 degrees (P < 0.001), who had more than 5 degrees of curve progression (P < 0. 05), or who had undergone posterior spinal fusion (P < 0.001). CONCLUSIONS: These long-term data confirm that the Boston brace when used 18 or more hours per day is effective in preventing progression of large curves at a mean of 9.8 years after bracing is discontinued.

New insights into neural injury, repair, and adaptation in visceral afferents and the enteric nervous system.

In this brief review, we focus on some of the proposed mechanisms of injury in peripheral visceral afferents (sensory) pathways and the enteric nervous system, including the interstitial cells of Cajal. Injury involving afferent neurons is discussed because of the relevance of these neurons to the pathophysiology of pain syndromes. The effect of various noxious stimuli on sensory and enteric neural function is examined. Finally, we discuss recent data on the role of autoimmune antibodies in neuronal injury in systemic diseases, such as diabetes mellitus and the Lambert-Eaton myasthenic syndrome. Neither central nervous system manifestations of peripheral nerve injury nor functional bowel disorders are addressed in this review. An improved understanding of the pathophysiology of peripheral neuronal dysfunction will probably result in new treatment strategies for a broad range of gastrointestinal disorders, including constipation, pseudo-obstruction, ileus, and inflammatory bowel disorders.

Serum from patients with type 2 diabetes with neuropathy induces complement-independent, calcium-dependent apoptosis in cultured neuronal cells.

We hypothesized that sera from type 2 diabetic patients with neuropathy contains an autoimmune immunoglobulin that promotes complement-independent, calcium-dependent apoptosis in neuronal cell lines. Neuronal cells were cultured in the presence of complement-inactivated sera obtained from patients with type 2 diabetes with and without neuropathy and healthy adult control patients. Serum from diabetic patients with neuropathy was associated with a significantly greater induction of apoptosis, compared to serum from diabetic patients without neuropathy and controls. In the presence of calcium channel antagonists, induction of apoptosis was reduced by approximately 50%. Pretreatment of neuronal cells with serum from diabetic patients with neuropathy was associated with a significant increase in elevated K+-evoked cytosolic calcium concentration. Serum-induced enhancement in cytosolic calcium and calcium current density was blocked by treatment with trypsin and filtration of the serum using a 100,000-kd molecular weight filter. Treatment with an anti-human IgG antibody was associated with intense fluorescence on the surface of neuronal cells exposed to sera from patients with type 2 diabetes mellitus with neuropathy. We conclude that sera from type 2 diabetic patients with neuropathy contains an autoimmune immunoglobulin that induces complement-independent, calcium-dependent apoptosis in neuronal cells.

Serum from diabetic BB/W rats enhances calcium currents in primary sensory neurons.

We examined the hypothesis that exposure of nondiabetic rat dorsal root ganglion (DRG) neurons to sera from diabetic BB/W rats would produce an increase in calcium currents associated with impaired regulation of the inhibitory G protein-calcium channel complex. Acutely dissociated rat DRGs were incubated for 18-24 h in medium supplemented with sera (10% vol/vol) from either diabetic rats with neuropathy or age-matched, nondiabetic controls. Exposure of DRG neurons to sera from diabetic BB/W rats resulted in a surface membrane immunofluorescence pattern when treated with an anti-rat light-chain antibody that was not observed in neurons exposed to control sera. Calcium current density (IDCa) was assessed with the use of the whole cell variation of the patch-clamp technique. IDCa in neurons exposed to diabetic sera was significantly increased compared with neurons exposed to control sera. Guanine nucleotide-binding (G) protein regulation of calcium channel function was examined with the use of a two-pulse "facilitation" or IDCa enhancement protocol in the presence of activators [guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)] or antagonists [guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) and pertussis toxin (PTX)] of G protein function. Facilitation was significantly decreased in neurons exposed to diabetic sera. Intracellular diffusion of neurons with GDP beta s blocked facilitation, whereas dialysis with GTP gamma s increased facilitation to a similar magnitude in neurons exposed to either diabetic or control sera. Treatment with PTX resulted in a significant increase in IDCa and approximately 50% decrease in facilitation in neurons treated with control sera but no significant changes in neurons exposed to diabetic sera. We conclude that serum from diabetic BB/W rats with neuropathy contains an autoimmune immunoglobulin that impairs regulation of the inhibitory G protein-calcium channel complex, resulting in enhanced calcium influx. Regulation of the inhibitory G protein-calcium channel complex involves PTX-sensitive and -insensitive G proteins.

Neural injury, repair and adaptation in the GI tract. I. New insights into neuronal injury: a cautionary tale.

Understanding of the pathophysiology of neuronal injury has advanced remarkably in the last decade. This largely reflects the burgeoning application of molecular techniques to neuronal cell biology. Although there is certainly no consensus hypothesis that explains all aspects of neuronal injury, a number of interesting observations have been published. In this brief review, we examine mechanisms that appear to contribute to the pathophysiology of neuronal injury, including altered Ca2+ signaling, activation of the protease cascades coupled to apoptosis, and mitochondrial deenergization associated with release of cytochrome c, production of free radicals, and oxidative injury. Finally, evidence for neuroprotective mechanisms that may ameliorate cell injury and/or death are reviewed. Little information has been published regarding the mechanisms that mediate injury in the enteric nervous system, necessitating a focus on models outside the gastrointestinal (GI) tract, which may provide insights into enteric nervous system injury.

Dynorphin A-mediated reduction in multiple calcium currents involves a G(o) alpha-subtype G protein in rat primary afferent neurons.

We examined the effect of antisera directed at specific G-protein subtype(s) on dynorphin A (Dyn A)-mediated reduction of calcium currents in rat dorsal root ganglia (DRG) neurons. Whole cell patch-clamp recordings were performed on acutely dissociated neurons. Dyn A (1 microM)-mediated decrease in calcium currents was inhibited > 90% by the preferential kappa-receptor antagonist norbinaltorphimine. Dyn A (300-1,000 nM)-mediated reduction in calcium currents was examined during intracellular administration of antisera directed against specific regions of G(o) alpha, G(i) 1 alpha/G(1) 2 alpha, and G(i) 3 alpha subunits. Intracellular dialysis with an antiserum specific for G(o) alpha for 20 min decreased calcium current inhibition by Dyn A (1 microM) in 13 of 15 neurons by an average of 75%. Dialysis with nonimmune serum did not affect Dyn A's action to reduce calcium currents. Intracellular dialysis with either anti-G(i) 1 alpha/G(i) 2 alpha or anti-G(i) 3 alpha antisera did not affect Dyn A-induced changes in calcium currents. In the presence of the N-type calcium channel antagonist omega-conotoxin GVIA, the P-type calcium channel antagonist omega-Aga IVA, and omega-Aga MVIIC applied subsequent to the other toxins, the effect of Dyn A to reduce calcium currents was inhibited by 52, 28, and 16%, respectively. The L channel antagonist nifedipine did not affect the ability to Dyn A to inhibit calcium currents. These results suggest that in rat DRG neurons coupling of kappa-opioid receptors to multiple transient, high-threshold calcium currents involves the G(o) alpha subclass of G proteins.

Failure of nimodipine to prevent or correct the long-term nerve conduction defect and increased neuronal Ca(2+)-currents in the diabetic BB/W-rat.

It has been suggested that L-type Ca2+ channel antagonists exert a beneficial effect on nerve conduction velocity (NCV) slowing in short-term experimental diabetic neuropathy. We examined the effects of long-term treatment with the L-channel blocker, nimodipine, on two aspects of neuronal function previously documented to be abnormal in the spontaneously diabetic BB/W-rat: nerve conduction velocity and calcium influx in dorsal root ganglion (DRG) neurons. Treatment with 20 mg/kg nimodipine i.p. every 48 h from onset of diabetes for 6 months led to a transient, non-significant (30%) improvement in NCV. Intervention with the same regimen from 3 to 6 months of diabetes had no corrective effect on the already established NCV defect. Voltage activated calcium currents were recorded in isolated DRG neurons from nimodipine-treated and untreated diabetic and non-diabetic age-matched BB/W control rats. The peak high-threshold calcium current density (IDCa, pA/pF) was significantly larger in non-treated diabetic rats compared with control rats (157 +/- 12 vs. 66 +/- 5.5 (P < or = 0.05)). Long-term treatment with nimodipine was associated with a non-significant (28%) decrease (112 +/- 29) in the IDCa compared with non-treated diabetic rats. We conclude that L-channel mediated perturbations of cytosolic Ca2+ levels are only of minor pathophysiologic significance in the development of chronic diabetic neuropathy.

Opiate-mediated inhibition of calcium signaling is decreased in dorsal root ganglion neurons from the diabetic BB/W rat.

The effect of diabetes mellitus on opiate-mediated inhibition of calcium current density (I(D Ca) [pA pF-1]) and cytosolic calcium response ([Ca2+]i nM) to depolarization with elevated KCl and capsaicin was assessed. Experiments were performed on isolated, acutely dissociated dorsal root ganglion (DRG) neurons from diabetic, BioBreeding/Worcester (BB/W) rats and age-matched control animals. Sciatic nerve conduction velocity was significantly decreased in diabetic animals compared to controls. Mean I(DCa) and [Ca2+]i responses to capsaicin and elevated KCl recorded in DRGs from diabetic animals were significantly larger than those recorded in DRG neurons from controls. In neurons from diabetic animals, the opiate agonist dynorphin A (Dyn A; 1, 3, and 5 microM) had significantly less inhibitory effect on I(D Ca) and KCl-induced [Ca2+]i responses compared to controls. Omega-conotoxin GVIA (omega-CgTX; 10 microM) and pertussis toxin (PTX; 250 ng ml-1) abolished Dyn A-mediated inhibition of I(DCa) and [Ca2+]i in control and diabetic neurons, suggesting that Dyn A modulated predominantly N-type calcium channels coupled to opiate receptors via PTX-sensitive (Gi/o) inhibitory G proteins. These results suggest that opiate-mediated regulation of PTX-sensitive, G protein-coupled calcium channels is diminished in diabetes and that this correlates with impaired regulation of cytosolic calcium.

Voltage-dependent calcium currents are enhanced in dorsal root ganglion neurones from the Bio Bred/Worchester diabetic rat.

1. Whole-cell, high-threshold, voltage-dependent calcium currents (ICa) were enhanced in acutely dissociated, capsaicin-sensitive dorsal root ganglion neurones from diabetic Bio Bred/Worchester (BB/W) rats, compared with those from age-matched, non-diabetic controls. The magnitude of the enhancement increased with the duration of diabetes, and reached significance at diabetic durations of 6 months (diabetic: 6.3 +/- 0.4 nA; current density (CD), 157 +/- 12 pA pF-1; means +/- S.E.M., n = 9, P < 0.01; control: 3.9 +/- 0.6 nA; CD, 116 +/- 11 pA pF-1; n = 18) and 8 months (diabetic: 7.6 +/- 0.4 nA; CD, 177 +/- 25 pA pF-1; n = 11, P < 0.005; control: 5.1 +/- 0.5 nA; CD, 111 +/- 26 pA pF-1; n = 15). Low-threshold, voltage-dependent ICa were also enhanced in neurones from animals diabetic for 8 months (diabetic: 2.5 +/- 0.7 nA, n = 4, P < 0.05; control: 0.7 +/- 0.5 nA, n = 6). 2. The ICa enhancement was prevented by long-term treatment of diabetic animals with an aldose reductase inhibitor (ARI; peak ICa at 6 months: 4.41 +/- 0.48 nA, n = 2; at 8 months: 4.32 +/- 0.60 nA, n = 9). 3. The ICa enhancement was not due to a shift in the voltage dependence of either the current-voltage relationship or steady-state inactivation. 4. The L channel antagonist nifedipine and preferential N channel antagonist omega-conotoxin GVIA (omega-CgTX) caused a greater inhibition of high-threshold ICa in diabetic neurones compared with controls (nifedipine: control: 25 +/- 3%, n = 26; diabetic: 36 +/- 7%, n = 11; omega-CgTX: control: 40 +/- 4%, n = 21; diabetic: 50 +/- 7%, n = 7). Diabetic neurones also demonstrated a significantly greater residual current (2.44 +/- 0.34 nA, n = 7) in the presence of both antagonists vs. controls (1.28 +/- 0.30 nA, n = 8, P < 0.05), suggesting that N-, L- and additional non-N-, non-L-type high-threshold ICa were enhanced.

Bradykinin (Bk) increases cytosolic calcium in cultured rat myenteric neurons via Bk-2 type receptors coupled to mobilization of extracellular and intracellular sources of calcium: evidence that calcium influx is prostaglandin dependent.

We examined the effect of bradykinin (Bk) on cytosolic calcium ([Ca++]i) in cultured rat ileal myenteric neurons. The receptor subtype(s) and calcium pool(s), e.g., extracellular and/or intracellular calcium that mediate Bk's effect on [Ca++]i in myenteric neurons, have not been reported. Superfusion with Bk (10 nM) increased [Ca++]i by 96 +/- 10 nM over basal levels in approximately 80% of the neurons tested that were not affected by a Bk-1 receptor antagonist but were inhibited 72% by a Bk-2 receptor antagonist. The Bk-generated increase in [Ca++]i was reduced by 45% and 52% of control response in calcium-free buffer and indomethacin, respectively, supporting involvement of extracellular and intracellular pools of calcium and mediation, in part, by a prostaglandin-dependent pathway. Bk increased [Ca2++]i by 54 +/- 6 nM in calcium-free buffer that was indomethacin insensitive, suggesting that Bk stimulation of extracellular calcium influx was mediated by a prostaglandin-dependent pathway. Bk (1 microM) increased tissue prostaglandin E2 (PGE2) production by 62% over basal levels in isolated rat ileal myenteric ganglia. Finally, superfusion with PGE2 (10 microM) increased [Ca++]i by 105 +/- 16 nM over basal levels that were blocked in calcium-free buffer. In summary, our studies suggest that cultured rat ileal myenteric neurons express the Bk-2 receptor subtype that is coupled to mobilization of extracellular and intracellular pools of calcium. Bk stimulates influx of extracellular calcium via a prostaglandin-dependent pathway.