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.     

Increased epinephrine-induced cAMP response in severely diabetic BB/W rat liver

The effect of prolonged diabetes on epinephrine-induced adenosine 3',5'-monophosphate (cAMP) response in the liver was examined in diabetes-prone BB/W rats. Basal and 1 microM epinephrine-induced cAMP release from isolated perfused liver was similar in non-diabetic and diabetic BB/W rats with preserved adipose tissue. In adipose tissue-absent diabetic rats losing intra- and retro-peritoneal adipose tissue completely, both basal and 1 microM epinephrine-induced cAMP release from the liver were enhanced (P<0.01, each case). Plasma epinephrine and norepinephrine were similar in non-diabetic, adipose tissue-preserved and -absent diabetic BB/W rats. The plasma free thyroxine level was similar in non-diabetic and adipose tissue-preserved diabetic BB/W rats, but was lower in adipose tissue-absent diabetic BB/W rats than in non-diabetic rats (P<0.01), but the frequency of lymphocytic thyroiditis was similar in these three groups, although plasma corticosterone was lower in adipose tissue-preserved diabetic BB/W rats (P<0.05) and the lowest in adipose tissue-absent diabetic BB/W rats (P<0.01). Lymphocytic infiltration was not observed in the adrenal or pituitary glands in any group. Plasma total protein and albumin were low in adipose tissue-absent diabetic BB/W rats (P<0.01, each case). In adipose tissue-absent diabetic BB/W rats, liver dysfunction and hepatomegaly, but no apparent histological change in the liver, were observed. Plasma glucose was higher (P<0.01) and plasma insulin lower (P<0.05) in adipose tissue-absent diabetic BB/W rats than in adipose tissue-preserved diabetic BB/W rats. In conclusion, epinephrine-induced cAMP response in the liver was enhanced only in adipose tissue-absent diabetic BB/W rats. Denervation supersensitivity was not likely to be responsible for the enhanced beta-adrenergic response. The observed reductions in plasma thyroxine and corticosterone seemed to result from severe diabetes. Although the severity of diabetes can vary continuously, severe diabetes with loss of adipose tissue appeared to cause significant changes in the metabolism and enhanced beta-adrenergic response in the liver.     

Facilitation of L-type calcium current in thalamic neurons

We have studied facilitation of the L-type calcium current in neurons acutely isolated from the ventrobasal nucleus of the rat thalamus. Currents were recorded after pretreatment with 1 microM omega-conotoxin GVIA and 5 microM omega-conotoxin MVIIC, to better isolate L-current. Long, strong depolarizations induced slow tail currents at negative voltages, but did not affect currents at voltages where channels were strongly activated. The initial peak tail current was not measurably increased. The time course of recovery from facilitation paralleled the time course of the tail current, indicating that facilitation does not outlast channel closing. The kinase inhibitors staurosporine and H-7 and the phosphatase inhibitor okadaic acid had no significant effect on L-current facilitation compared with control, but facilitation was greater with H-7 than with okadaic acid. The guanosine 5'-triphosphate (GTP) analogs GTP-gamma-S and GDP-beta-S did not affect facilitation. We conclude that L-current facilitation in thalamic neurons does not result from Ser/Thr phosphorylation, although phosphorylation may modulate facilitation. This form of facilitation differs kinetically and pharmacologically from facilitation induced by activation of G protein-coupled receptors.     

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.     

Inhibition of calcium currents in rat colon sensory neurons by K- but not mu- or delta-opioids

Inhibition of calcium currents in rat colon sensory neurons by kappa- but not mu- or delta-opioids. J. Neurophysiol. 80: 3112-3119, 1998. We previously reported that kappa-, but not mu- or delta-opioid receptor agonists (ORAs) have selective, potentially useful peripheral analgesic effects in visceral pain. To evaluate one potential site and mechanism by which these effects are produced, we studied opioid effects on high-voltage activated (HVA) Ca2+ currents in identified (Di-I) pelvic nerve sensory neurons from the S1 dorsal root ganglion (DRG). Results were compared with opioid effects on cutaneous neurons from L5 or L6 DRG. Di-I-labeled DRG cells were voltage clamped (perforated whole cell patch clamp), and HVA Ca2+ currents were evoked by depolarizing 240-ms test pulses to +10 mV from a holding potential of -60 mV. Neither mu-ORAs (morphine, 10(-6 )M, n = 16; [D-Ala2, N-Me-Phe4, Gly-ol5] enkephalin, 10(-6 )M, n = 12) nor delta-ORAs ([D-Pen2, D-Pen5] enkephalin, 10(-7 )M, n = 16; SNC-80, 10(-7 )M, n = 7) affected HVA Ca2+ currents in colon sensory neurons. In contrast, the kappa-ORAs U50, 488 (10(-6 )M), bremazocine (10(-6)M), and nalBzoH (10(-6 )M) significantly attenuated HVA Ca2+ currents in colon sensory neurons; effects on cutaneous sensory neurons were variable. A nonreceptor selective concentration of naloxone (10(-5 )M) and nor-BNI (10(-6 )M), a selective kappa-opioid receptor antagonist, reversed the inhibitory effect of kappa-ORAs. In the presence of N-, P-, or Q-, but not L-type Ca2+ channel antagonists, the effect of U50,488 on HVA Ca2+ currents was significantly reduced. Pretreatment with pertussis toxin (PTX) prevented the inhibition by U50,488. These results suggest that kappa-opioid receptors are coupled to multiple HVA Ca2+ channels in colon sensory neurons by a PTX-sensitive G protein pathway. We conclude that inhibition of Ca2+ channel function likely contributes in part to the peripheral analgesic action of kappa-ORAs in visceral nociception.     

High-voltage-activated calcium currents in neurons acutely isolated from the ventrobasal nucleus of the rat thalamus

We studied the high-voltage-activated (HVA) calcium currents in cells isolated from the ventrobasal nucleus of the rat thalamus with the use of the whole cell patch-clamp technique. Low-voltage-activated current was inactivated by the use of long voltage steps or 100-ms prepulses to -20 mV. We used channel blocking agents to characterize the currents that make up the HVA current. The dihydropyridine (DHP) antagonist nimodipine (5 microM) reversibly blocked 33 +/- 1% (mean +/- SE), and omega-conotoxin GVIA (1 microM) irreversibly blocked 25 +/- 5%. The current resistant to DHPs and omega-conotoxin GVIA was inhibited almost completely by omega-conotoxin MVIIC (90 +/- 5% at 3-5 microM) and was partially inhibited by omega-agatoxin IVA (54 +/- 4% block at 1 microM). We conclude that there are at least four main HVA currents in thalamic neurons: N current, L current, and two omega-conotoxin MVIIC-sensitive currents that differ in their sensitivity to omega-agatoxin IVA. We also examined modulation of HVA currents by strong depolarization and by G protein activation. Long (approximately 1 s), strong depolarizations elicited large, slowly deactivating tail currents, which were sensitive to DHP antagonists. With guanosine 5'-0-(3-thiotriphosphate) (GTP-gamma-S) in the intracellular solution, brief (approximately 20 ms), strong depolarization produced a voltage-dependent facilitation of the current (44 +/- 5%), compared with cells with GTP (22 +/- 7%) or guanosine 5'-O-(2-thiodiphosphate) (7 +/- 4%). However, the HVA current was inhibited only weakly by 100 microM acetylcholine (8 +/- 4%). Effects of the gamma-aminobutyric acid-B agonist baclofen were variable (3-39% inhibition, n = 12, at 10-50 microM).     

Glycerol-3-phosphate transport in Haemophilus influenzae: cloning, sequencing, and transcription analysis of the glpT gene

Dorsal root ganglion (DRG) sensory neurons are particularly vulnerable to diabetes mellitus. There is evidence that the disease decreases both circulating and retrogradely transported neurotrophic factors that are essential to the normal maintenance and function of these cells. A substantive loss of trophic support should cause DRG neurons to respond as though they were axotomized and, like an axotomy, cause significant changes in cytoskeletal gene expression within these cells. Such changes might contribute to the deficits in sensory neuronal function that characterize diabetic neuropathy. The current study used quantitative in situ hybridization to test the hypothesis that streptozotocin-induced diabetes, like an axotomy, increases class III beta-tubulin gene expression and decreases neurofilament 68-kDa gene expression in lumbar DRG neurons. In animals that had been diabetic for 8 weeks with mean blood glucose levels of 340 mg/dl, lumbar DRG class III beta-tubulin mRNA mean steady-state levels were twofold higher than those in age-matched nondiabetic controls. Moreover, in the same animals, diabetes decreased lumbar DRG 68-kDa neurofilament mRNA mean steady-state levels by more than half. These data show that diabetes causes changes in primary sensory neuronal cytoskeletal gene expression that mimic those caused by axotomy. Moreover, they support the idea that a loss of neurotrophic support contributes to the pathogenesis of diabetic neuropathy.     

Modulation of high-voltage activated Ca2+ channels in the rat periaqueductal gray neurons by mu-type opioid agonist

The effect of mu-type opioid receptor agonist, D-Ala2,N-MePhe4,Gly5-ol-enkephalin (DAMGO), on high-voltage-activated (HVA) Ca2+ channels in the dissociated rat periaqueductal gray (PAG) neurons was investigated by the use of nystatin-perforated patch recording mode under voltage-clamp condition. Among 188 PAG neurons tested, the HVA Ca2+ channels of 38 neurons (32%) were inhibited by DAMGO (DAMGO-sensitive cells), and the other 80 neurons (68%) were not affected by DAMGO (DAMGO-insensitive cells). The N-, P-, L-, Q-, and R-type Ca2+ channel components in DAMGO-insensitive cells shared 26.9, 37.1, 22.3, 7.9, and 5.8%, respectively, of the total Ca2+ channel current. The channel components of DAMGO-sensitive cells were 45.6, 25.7, 21.7, 4.6, and 2.4%, respectively. The HVA Ca2+ current of DAMGO-sensitive neurons was inhibited by DAMGO in a concentration-, time-, and voltage-dependent manner. Application of omega-conotoxin-GVIA occluded the inhibitory effect of DAMGO approximately 70%. So, HVA Ca2+ channels inhibited by DAMGO were mainly the N-type Ca2+ channels. The inhibitory effect of DAMGO on HVA Ca2+ channels was prevented almost completely by the pretreatment of pertussis toxin (PTX) for 8-10 h, suggesting that DAMGO modulation on N-type Ca2+ channels in rat PAG neurons is mediated by PTX-sensitive G proteins. These results indicate that mu-type opioid receptor modulates N-type HVA Ca2+ channels via PTX-sensitive G proteins in PAG neurons of rats.     

The human FGF-8 gene localizes on chromosome 10q24 and is subjected to induction by androgen in breast cancer cells

Using a syngeneic Wistar rat model we have shown that the Wistar rat thyroid (WRT) cell line causes significant and specific proliferation of lymph node T cells from normal Wistar rats, and of splenic T cells from a thyroiditis prone line of BB/W rats, when cultured in the presence of irradiated feeder cells. These T cell responses were associated with a marked increase in the number of CD8+ T cells. However, using normal Wistar rat T cells which had been previously exposed to WRT cells, rested and then re-exposed to WRT cells as antigen, we consistently found that the T cell population had been rendered unreactive, or anergic, to further thyroid cell stimulation. However, if recombinant rat IL-2 was added to the cultures, then T cell responsivity was seen on re-exposure to WRT cells. The lymphopenic BB/W rat also had T cells which showed a primary T cell response to the WRT cell line accompanied by a marked increase in CD8+ T cells. In contrast to the Wistar rat T cells, the BB/W T cells retained a proliferative responsiveness to WRT cells on re-exposure although such responsiveness could also be markedly enhanced with IL-2. These data suggested that antigen-mediated inhibitory signals were induced in normal Wistar rat T cells by the syngeneic WRT cell line, independent of the presence of co-stimulatory molecules. Furthermore, the thyroiditis prone BB/W rat T cells appeared to be less responsive to such anergy induction, perhaps contributing to their susceptibility to autoimmune thyroid disease.     

Spinal morphine/clonidine antinociceptive synergism: involvement of G proteins and N-type voltage-dependent calcium channels

When morphine and clonidine are coadministered into the spinal cord (intrathecally) the resulting antinociception is greater than would be expected if the drug responses were additive; thus, a synergistic interaction. The mechanism for this synergistic interaction was investigated using agents which alter calcium channel function and G protein function. Drugs were administered intrathecally to mice and antinociception was measured using the tail flick test. The L-type calcium channel antagonists nifedipine (15 micrograms) and verapamil (15 micrograms) and the N-type antagonist omega-conotoxin GVIA (3 and 30 ng) decreased ED50 values for both morphine and clonidine three-to five-fold. The L-type calcium channel activator Bay K 8644 had a biphasic effect; 1.7 ng increased, although 170 ng decreased, morphine and clonidine ED50 values. None of the calcium channel modifiers affected the morphine/clonidine synergism. In mice pretreated with pertussis toxin (PTX, one, 10-ng dose 21 days previously), the morphine ED50 value increased two-fold, although the clonidine ED50 value was not changed. PTX pretreatment did not alter the morphine/clonidine synergism. Also, in PTX-pretreated mice, nifedipine and 1.7 ng Bay K 8644 did not alter the morphine/clonidine synergism. However, in PTX-pretreated animals omega-conotoxin GVIA (3 ng) changed the morphine/clonidine synergism to an additive interaction. Thus, both N-type calcium channels and PTX-sensitive G proteins are likely involved in spinal morphine/clonidine synergism.     

Vascular endothelial growth factor and severity of nonproliferative diabetic retinopathy mediate retinal hemodynamics in vivo: a potential role for vascular endothelial growth factor in the progression of nonproliferative diabetic retinopathy

PURPOSE: To determine the effect of vascular endothelial growth factor and retinopathy level on retinal hemodynamics in nondiabetic and diabetic rats and to evaluate retinal hemodynamics in nondiabetic and diabetic patients. METHODS: Forty-eight diabetic and 22 nondiabetic patients had their diabetic retinopathy levels determined from fundus photographs according to Early Treatment Diabetic Retinopathy Study (ETDRS). Fluorescein angiograms were recorded from the left eye by video fluorescein angiography. Retinal blood flow was calculated from the digitized angiograms. Human recombinant vascular endothelial growth factor or vehicle alone was injected intravitreally into 13 nondiabetic and 11 diabetic rats. RESULTS: Retinal blood flow decreased 33% in patients with ETDRS retinopathy level 10 compared with control patients (P = .001) and increased sequentially in more advanced stages of retinopathy, with a strong correlation between retinal blood flow and retinopathy level (r2 = 0.434, P = .001). In the diabetic rats, retinal blood flow was decreased 35.6% (P = .01). Vascular endothelial growth factor maximally increased retinal blood flow by 36.1% in nondiabetic rats after 25 minutes (P = .001) and by 73.7% in diabetic rats after only 5 minutes (P = .01) and caused a greater response in diabetic than in nondiabetic rats. CONCLUSIONS: Retinal blood flow increases with advancing nonproliferative diabetic retinopathy in humans, and diabetes accentuates the vascular endothelial growth factor-induced increase in retinal blood flow and venous dilation in rats. Vascular endothelial growth factor may contribute to the changes in retinal hemodynamics and morphology observed in early diabetic retinopathy.     

Lipocortin-1 and the control of arachidonic acid release in cell signalling. Glucocorticoids (changed from glucorticoids) inhibit G protein-dependent activation of cPLA2 activity

In pre-labelled A549 cells epidermal growth factor (EGF) (10 nM) stimulates the release of [5,6,8,9,11,12,14,15-3H(N)]-arachidonic acid (3H-AA) by approximately 70%. Increasing Ca2+i with thapsigargin (50 nM) stimulates 3H-AA release by approximately 120%. However, the combined use of these two agents results in a synergistic stimulation of 3H-AA release by over 700%. The EGF stimulated release is sensitive to pertussis toxin (10 ng/mL) and guanosine 5'-O-(2-thiodiphosphate) suggesting a G protein-mediated event. This is supported by the fact that the G protein activators AlF-4 and guanosine 5'-O-(2-thiotriphosphate) both stimulate 3H-AA release. The stimulation of 3H-AA release by both EGF or direct G protein activation is completely blocked following pre-treatment for 3 hr with 1 nM dexamethasone. This effect is reversed with a neutralizing antibody to lipocortin-1 (1 microgram/mL) suggesting that this protein mediates the inhibitory effects of glucocorticoids on agonist activated 3H-AA release. Thapsigargin stimulation of 3H-AA release is insensitive to dexamethasone treatment. A peptide fragment from the N-terminus of lipocortin-1-Lc13-25 (20-200 micrograms/mL) mimics the effect of glucocorticoid in suppressing both EGF and G protein activated 3H-AA release. A peptide with Me-Tyr substituting Tyr21 is much reduced in activity suggesting that the presence of this residue is essential. As peptide Lc13-25 is not derived from the Ca2+/phospholipid binding domain of the native protein then sequestration of phospholipid substrate for PLA2 remains an unlikely mechanism of action for this peptide.     

Muscarinic receptor activation modulates Ca2+ channels in rat intracardiac neurons via a PTX- and voltage-sensitive pathway

With use of the whole cell patch-clamp technique, effects of the potent muscarinic agonist oxotremorine methiodide (oxo-M) on voltage-activated Ca2+ channel currents were investigated in acutely dissociated adult rat intracardiac neurons. In all tested neurons oxo-M reversibly inhibited the peak Ba2+ current. Inhibition of the peak Ba2+ current by oxo-M was associated with slowing of activation kinetics and was concentration dependent. The concentration of oxo-M necessary to produce a half-maximal inhibition of current and the maximal inhibition were 40.8 nM and 75.9%, respectively. Inhibitory effect of oxo-M was completely abolished by atropine. Among different muscarinic receptor antagonists, methoctramine (100 and 300 nM) significantly antagonized the current inhibition by oxo-M, with a negative logarithm of dissociation constant of 8.3 in adult rat intracardiac neurons. Internal dialysis of neurons with guanosine 5'-(thio)triphosphate (GTPgammaS, 0.5 mM) could mimic the muscarinic inhibition of the peak Ba2+ current and significantly occlude inhibitory effects of oxo-M. In addition, the internal dialysis of guanosine-5'-O-(2-thiodiphosphate) (GDPbetaS, 2 mM) also significantly reduced the muscarinic inhibition of the peak Ba2+ current by oxo-M. Inhibitory effects of oxo-M were significantly abolished by pertussis toxin (PTX, 200 and 400 ng/ml) but not by cholera toxin (400 ng/ml). Furthermore, the bath application of N-ethylmaleimide (50 microM) significantly reduced the inhibition of the peak Ba2+ current by oxo-M. The oxo-M shifted the activation curve derived from measurments of tail currents toward more positive potentials. A strong conditioning prepulse to +100 mV significantly relieved the muscarinic inhibition of peak Ba2+ currents by oxo-M and the GTPgammaS-induced current inhibition. In a series of experiments, changes in intracellular concentration of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid and protein kinase activities failed to mimic or occlude the current inhibition by oxo-M. The dihydropyridine antagonist nifedipine (10 microM) was not able to occlude any of the inhibitory effects of oxo-M, and oxo-M (3 microM) failed to reduce the slow tail currents induced by the L-type agonist methyl 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate (FPL 64176; 2 microM). However, omega-conotoxin (omega-CgTX) GVIA (1 microM) significantly occluded the muscarinic inhibition of the Ba2+ currents. In the presence of omega-CgTX GVIA (1 microM) and nifedipine (10 microM), oxo-M could further inhibit approximately 20% of the total Ca2+ current. After complete removal of N-, Q-, and L-type currents with use of omega-CgTX GVIA, omega-agatoxin IVA, and nifedipine, 70% of the R-type current (approximately 6-7% of the total current) was inhibited by oxo-M (3 microM). In conclusion, the M2 muscarinic receptor activation selectively inhibits N-, Q-, and R-type Ca2+ channel currents, sparing L-type Ca2+ channel currents mainly via a PTX- and voltage-sensitive pathway in adult rat intracardiac neurons.     

Role of intracellular calcium in modification of mu and delta opioid receptor-mediated antinociception by diabetes in mice

We examined the effects of calcium modulators on mu and delta opioid receptor agonist-induced antinociception in both diabetic and nondiabetic mice. In nondiabetic mice, intracerebroventricular (i.c. v.) pretreatment with calcium and thapsigargin, which increase intracellular calcium, reduced [D-Ala2,N-MePhe4,Gly-ol5]-enkephalin (DAMGO)-induced antinociception by shifting its dose-response curve to the right. However, in diabetic mice i.c.v. pretreatment with calcium and thapsigargin did not affect DAMGO-induced antinociception. In contrast i.c.v. administration of agents that decrease intracellular calcium, such as EGTA and ryanodine, enhanced DAMGO-induced antinociception in both diabetic and nondiabetic mice. In contrast with DAMGO i.c.v. pretreatment with calcium and thapsigargin enhanced (-)-TAN67-induced antinociception in nondiabetic mice by shifting its dose-response curve to the left. However, (-)-TAN67-induced antinociception in diabetic mice was not affected by pretreatment with calcium or thapsigargin. Moreover i.c. v. pretreatment with EGTA, but not with ryanodine, reduced (-)-TAN67-induced antinociception in nondiabetic mice. In diabetic mice i.c.v. pretreatment with both EGTA and ryanodine reduced (-)-TAN67-induced antinociception. These results suggest that cytosolic calcium has different effects on mu and delta opioid receptor agonist-induced antinociception. Further, these results suggest that the modification of mu and delta opioid receptor agonist-induced antinociception by diabetes in mice may be due to increased levels of intracellular calcium.     

M4 muscarinic receptor activation modulates calcium channel currents in rat intracardiac neurons

Modulation of high-voltage-activated Ca2+ channels by muscarinic receptor agonists was investigated in isolated parasympathetic neurons of neonatal rat intracardiac ganglia using the amphotericin B perforated-patch whole cell recording configuration of the patch-clamp technique. Focal application of the muscarinic agonists acetylcholine (ACh), muscarine, and oxotremorine-M to the voltage-clamped soma membrane reversibly depressed peak Ca2+ channel current amplitude. The dose-response relationship obtained for ACh-induced inhibition of Ba2+ current (IBa) exhibited a half-maximal inhibition at 6 nM. Maximal inhibition of IBa amplitude obtained with 100 microM ACh was approximately 75% compared with control at +10 mV. Muscarinic agonist-induced attenuation of Ca2+ channel currents was inhibited by the muscarinic receptor antagonists pirenzepine (/=30% at +90 mV in the presence of ACh, indicating a voltage-independent component to the muscarinic receptor-mediated inhibition. Both dihydropyridine- and omega-conotoxin GVIA-sensitive and -insensitive Ca2+ channels were inhibited by ACh, suggesting that the M4 muscarinic receptor is coupled to multiple Ca2+ channel subtypes in these neurons. Inhibition of IBa amplitude by muscarinic agonists was also observed after cell dialysis using the conventional whole cell recording configuration. However, internal perfusion of the cell with 100 microM guanosine 5'-O-(2-thiodiphosphate) trilithium salt (GDP-beta-S) or incubation of the neurons in Pertussis toxin (PTX) abolished the modulation of IBa by muscarinic receptor agonists, suggesting the involvement of a PTX-sensitive G-protein in the signal transduction pathway. Given that ACh is the principal neurotransmitter mediating vagal innervation of the heart, the presence of this inhibitory mechanism in postganglionic intracardiac neurons suggests that it may serve for negative feedback regulation.     

Impaired expression of atrial natriuretic peptide in diabetic rats with myocardial infarction

We evaluated the effects of myocardial infarction (MI) on the hemodynamics and the expression of atrial natriuretic peptide (ANP) mRNA in rats with streptozotocin-induced diabetes. Eight weeks after streptozotocin injection, the diabetic rats and age-matched nondiabetic controls underwent coronary artery ligation. One week later, the left ventricular end-diastolic pressure, systolic blood pressure, infarct size, and serum ANP levels did not differ significantly between the diabetic and nondiabetic rats. Compared with control animals without MI, the atrial ANP/beta-actin mRNA ratio in rats with MI was increased to 195% in diabetic animals and 213% in nondiabetic animals. In the left ventricle, however, the ANP/beta-actin mRNA ratio in diabetic animals with MI was increased to only 131% compared with control animals, whereas the corresponding increase in nondiabetic animals was 240% (p<0.05). Thus, the modulation of ANP mRNA expression after MI was impaired in the left ventricle, but not in the atria, of diabetic rats. A reduced myocardial expression of ANP could increase the morbidity and mortality associated with cardiovascular disorders in patients with diabetes.     

Renoprotective effect of enalapril in uninephrectomized spontaneously hypertensive rats with neonatal streptozotocin-induced diabetes

We compared the renoprotective effect between angiotensin-converting enzyme inhibitor, enalapril, and a dihydropyridine-type calcium channel blocker, nicardipine, in a severe form of renal injury in rats. Two-day-old spontaneously hypertensive rats (SHR) were injected with streptozotocin or vehicle as control. UNX was performed at 3 weeks of age, and enalapril or nicardipine was administered in drinking water from 7 weeks of age. Uninephrectomy (UNX) markedly exacerbated hypertension and renal injury in the nondiabetic and diabetic SHR. Enalapril and nicardipine comparably reduced blood pressure in UNX diabetic SHR. However, serum creatinine was significantly elevated in the nicardipine-treated group as compared with the enalapril-treated group at 24 weeks of age (nicardipine-treated group, 67 +/- 4 microM; enalapril-treated group, 49 +/- 3 microM; P < 0.01; untreated group 57 +/- 4 microM). Furthermore, the incidence of glomerular sclerosis was similar between untreated and nicardipine-treated groups, whereas it tended to be reduced in the enalapril-treated group. In a separate experiment of diabetic SHR without UNX, enalapril therapy significantly ameliorated hyperglycemia and albuminuria (P < 0.01). This study showed that a renoprotective effect was seen in enalapril but not in nicardipine in UNX diabetic SHR despite the comparable reduction of blood pressure. This suggests that enalapril may be more effective than nicardipine in delaying the progression of a severe form of diabetic nephropathy.     

Rat group I metabotropic glutamate receptors inhibit neuronal Ca2+ channels via multiple signal transduction pathways in HEK 293 cells

We have shown previously that metabotropic glutamate receptors with group I-like pharmacology couple to N-type and P/Q-type calcium channels in acutely isolated cortical neurons using G proteins most likely belonging to the Gi/Go subclass. To better understand the potential mechanisms forming the basis for group I mGluR modulation of voltage-gated calcium channels in the CNS, we have examined the ability of specific mGluRs to couple to neuronal N-type (alpha1B-1/alpha2delta/beta1b) and P/Q-type (alpha1A-2/alpha2delta/beta1b) voltage-gated calcium channels in an HEK 293 heterologous expression system. Using the whole cell patch-clamp technique where intracellular calcium is buffered to low levels, we have shown that group I receptors inhibit both N-type and P/Q-type calcium channels in a voltage-dependent fashion. Similar to our observations in cortical neurons, this voltage-dependent inhibition is mediated almost entirely by N-ethylmaleimide (NEM)-sensitive heterotrimeric G proteins, strongly suggesting that these receptors can use Gi/Go-like G proteins to couple to N-type and P/Q-type calcium channels. However, inconsistent with the apparent NEM sensitivity of group I modulation of calcium channels, modulation of N-type channels in group I mGluR-expressing cells was only partially sensitive to pertussis toxin (PTX), indicating the potential involvement of both PTX-sensitive and -resistant G proteins. The PTX-resistant modulation was voltage dependent and entirely resistant to NEM and cholera toxin. A time course of treatment with PTX revealed that this toxin caused group I receptors to slowly shift from using a primarily NEM-sensitive G protein to using a NEM-resistant form. The PTX-induced switch from NEM-sensitive to -resistant modulation was also dependent on protein synthesis, indicating some reliance on active cellular processes. In addition to these voltage-dependent pathways, perforated patch recordings on group I mGluR-expressing cells indicate that another slowly developing, calcium-dependent form of modulation for N-type channels may be seen when intracellular calcium is not highly buffered. We conclude that group I mGluRs can modulate neuronal Ca2+ channels using a variety of signal transduction pathways and propose that the relative contributions of different pathways may exemplify the diversity of responses mediated by these receptors in the CNS.     

Passive transfer of insulitis from the "BB" rat to the nude mouse

The "BB" rat spontaneously develops insulitis, and an insulin-dependent diabetic syndrome like that in man. Lymphocytes were isolated from blood and spleen of newly-detected "BB" diabetic rats and injected intraperitoneally (IP) into athymic nude mice. Of 72 mice receiving single injections 37% showed insulitis, with 13% of islets examined being affected, and mean intensity of 1.9 +/- 0.3 (on a scale of 0 to 3). In 12 mice receiving 3 separate injections of pooled blood and spleen lymphocytes, 58% showed insulitis, with 17% of islets affected, and mean intensity 2.5 +/- 0.3. Of 45 control mice either untreated, injected IP with saline, or injected with cells from nondiabetic control rats, only one showed mild insulitis. No random or post IP glucose hyperglycemia was observed. Thus, 1) passive transfer of insulitis has been achieved; 2) insulitis may be present without glucoregulatory disturbances; 3) the pancreatic B cell need not display abnormal membrane structure for it to be susceptible to involvement in the cell-mediated immune process; and 4) detailed studies are required to define the relationship of administered lymphocytes to the observed pathology.