Defective translocation of PKCepsilon in EtOH-induced inhibition of Mg2+ accumulation in rat hepatocytes.

BACKGROUND: Rats chronically fed ethanol for 3 weeks presented a marked decreased in total hepatic Mg(2+) content and required approximately 12 days to restore Mg(2+) homeostasis upon ethanol withdrawal. This study was aimed at investigating the mechanisms responsible for the EtOH-induced delay. METHODS: Hepatocytes from rats fed ethanol for 3 weeks (Lieber-De Carli diet-chronic model), rats re-fed a control diet for varying periods of time following ethanol withdrawal, and age-matched control rats fed a liquid or a pellet diet were used. As acute models, hepatocytes from control animals or HepG2 cells were exposed to varying doses of ethanol in vitro for 8 minutes. RESULTS: Hepatocytes from ethanol-fed rats presented a marked inhibition of Mg(2+) accumulation and a defective translocation of PKCepsilon to the cell membrane. Upon ethanol withdrawal, 12 days were necessary for PKCepsilon translocation and Mg(2+) accumulation to return to normal levels. Exposure of control hepatocytes or HepG2 cells to a dose of ethanol as low as 0.01% for 8 minutes was already sufficient to inhibit Mg(2+) accumulation and PKCepsilon translocation for more than 60 minutes. Also in this model, recovery of Mg(2+) accumulation was associated with restoration of PKCepsilon translocation. The use of specific antisense in HepG2 cells confirmed the involvement of PKCepsilon in modulating Mg(2+) accumulation. CONCLUSIONS: Translocation of PKCepsilon isoform to the hepatocyte membrane is essential for Mg(2+) accumulation to occur. Both acute and chronic ethanol administrations inhibit Mg(2+) accumulation by specifically altering PKCepsilon translocation to the cell membrane.

The serum of dysautonomia patients enhances proliferation and signaling in Schwann cells.

Disorders of the autonomic nervous system, or dysautonomias, affect a large segment of the population, especially women, and represent a diagnostic challenge. Identification of biomarkers for autonomic disorders, and the subsequent development of screening methods, would benefit diagnosis and symptom management. We studied the effect of sera from fifteen well-characterized dysautonomia patients (mean age 49+/-16 years, 10 females, 5 males) and ten control subjects (mean age 31+/-14 years, 5 females, 5 males) on the proliferation of cultured Schwann cells and activity of mitogen-activated protein kinases (MAPKs) in these cells. We correlated characteristics of patients with the effects on cell proliferation and signaling. Overall, we observed a significant increase in proliferation when Schwann cells were incubated with sera from female dysautonomia patients when compared to control subjects and male patients. Interestingly, removal of IgGs significantly reduced the proliferative effect of patient sera. We also observed significant activation of p38 MAPK following incubation with both male and female patient sera. These results suggest that patient sera contain factors that contribute to aberrant Schwann cell proliferation and signaling and may ultimately lead to autonomic nerve dysfunction. Our observations represent a promising first step in the identification of dysautonomia biomarkers.

Sulfasalazine blocks the development of tactile allodynia in diabetic rats.

OBJECTIVE: Diabetic neuropathy is manifested either by loss of nociception (painless syndrome) or by mechanical hyperalgesia and tactile allodynia (pain in response to nonpainful stimuli). While therapies with vasodilators or neurotrophins reverse some functional and metabolic abnormalities in diabetic nerves, they only partially ameliorate neuropathic pain. The reported link between nociception and targets of the anti-inflammatory drug sulfasalazine prompted us to investigate its effect on neuropathic pain in diabetes. RESEARCH DESIGN AND METHODS: We examined the effects of sulfasalazine, salicylates, and the poly(ADP-ribose) polymerase-1 inhibitor PJ34 on altered nociception in streptozotocin-induced diabetic rats. We also evaluated the levels of sulfasalazine targets in sciatic nerves and dorsal root ganglia (DRG) of treated animals. Finally, we analyzed the development of tactile allodynia in diabetic mice lacking expression of the sulfasalazine target nuclear factor-kappaB (NF-kappaB) p50. RESULTS: Sulfasalazine completely blocked the development of tactile allodynia in diabetic rats, whereas relatively minor effects were observed with other salicylates and PJ34. Along with the behavioral findings, sciatic nerves and DRG from sulfasalazine-treated diabetic rats displayed a decrease in NF-kappaB p50 expression compared with untreated diabetic animals. Importantly, the absence of tactile allodynia in diabetic NF-kappaB p50(-/-) mice supported a role for NF-kappaB in diabetic neuropathy. Sulfasalazine treatment also increased inosine levels in sciatic nerves of diabetic rats. CONCLUSIONS: The complete inhibition of tactile allodynia in experimental diabetes by sulfasalazine may stem from its ability to regulate both NF-kappaB and inosine. Sulfasalazine might be useful in the treatment of nociceptive alterations in diabetic patients.

Lack of insulin impairs Mg2+ homeostasis and transport in cardiac cells of streptozotocin-injected diabetic rats.

Serum and tissue Mg(2+) content are markedly decreased in diabetic patients and animals. At the tissue level, Mg(2+) loss progresses over time and affects predominantly heart, liver and skeletal muscles. In the present study, alterations in Mg(2+) homeostasis and transport in diabetic cardiac ventricular myocytes were evaluated. Cardiac tissue and isolated cardiac ventricular myocytes from diabetic animals displayed a decrease in total Mg(2+) content that affected all cellular compartments. This decrease was associated with a marked reduction in cellular protein and ATP content. Diabetic ventricular myocytes were unable to mobilize Mg(2+) following beta-adrenergic receptor stimulation or addition of cell permeant cyclic-AMP. Sarcolemma vesicles purified from diabetic animals, however, transported Mg(2+) normally as compared to vesicles from non-diabetic animals. Treatment of diabetic animals with exogenous insulin for 2 weeks restored ATP and protein levels as well as Mg(2+) homeostasis and transport to levels comparable to those observed in non-diabetic animals. These results suggest that in diabetic cardiac cells Mg(2+) homeostasis and extrusion via beta-adrenergic/cAMP signaling are markedly affected by the concomitant decrease in protein and ATP content. As Mg(2+) regulates numerous cellular enzymes and functions, including protein synthesis, these results provide a new rationale to interpret some aspects of the cardiac dysfunctions observed under diabetic conditions.

Reduced expression of endothelin B receptors and mechanical hyperalgesia in experimental chronic diabetes.

Diabetic neuropathy is one of the major complications of diabetes mellitus. Small nerve fibers degenerate early in the disease, leading to symptoms ranging from hyperalgesia to loss of pain and temperature sensation. However, the cellular and molecular mechanisms responsible for abnormal pain perception in diabetes have not been identified. Both type-A and type-B endothelin receptors (ETAR and ETBR, respectively) are present in sensory nerves and appear to regulate neuropathic and inflammatory pain. In this study, we compared the expression of endothelin receptors and nociceptive responses in normal and experimentally diabetic rats. Diabetic animals exhibited both an increase in the withdrawal responses to high threshold stimuli (mechanical hyperalgesia) and to light touch stimuli (tactile allodynia). Immunohistochemical and Western blot analysis revealed that diabetic rats have significantly reduced expression of ETBR in sciatic nerves, while no changes were observed in dorsal root ganglia (DRG). In contrast, the expression of ETAR in either sciatic nerves or DRG of diabetic rats was not altered. Importantly, ETBR-deficient transgenic rats showed alterations in pain perception similar to those observed in diabetic rats. These results suggest that changes in the expression of ETBR in peripheral nerve may contribute to the development of mechanical hyperalgesia and tactile allodynia in chronic diabetes.

Transient expression of hypoxia-inducible factor-1 alpha and target genes in peripheral nerves from diabetic rats.

Decreased blood flow is one of the earliest physiological changes observed after the onset of either clinical or experimental diabetes. The reduction in blood flow is believed to lead to nerve hypoxia, which in conjunction with other metabolic alterations and degenerative processes in different nerve compartments, results in the dysfunction known as diabetic neuropathy. The transcriptional regulator hypoxia-inducible factor-1 alpha (HIF-1alpha) accumulates rapidly under hypoxic conditions and modulates the expression of several target genes that protect tissues against ischemia and infarction. At present it is unclear whether diabetic nerve injury results from an abnormal response of HIF-1alpha and its protective target genes. In the present study we have analyzed the expression and activity of HIF-1alpha and its target genes in diabetic nerves as a first step to determine their possible contribution to the development or maintenance of diabetic neuropathy. We observed a transient increase in the expression of HIF-1alpha that peaked between 4 and 6 weeks and declined 8 weeks after induction of experimental diabetes in rats. The increase in HIF-1alpha in diabetic nerves coincided with a similarly transient increase in the expression of several HIF-1alpha target genes including vascular endothelial growth factor, lactate dehydrogenase and erythropoietin, which subsided 8-10 weeks after induction of diabetes. These results suggest that the transient activation of neurotrophic and angiogenic genes, as opposed to a more sustained effect in response to the chronic injury, may be responsible for the alterations in nerve function and regeneration that characterize the diabetic neuropathy.

Hyperglycemia triggers abnormal signaling and proliferative responses in Schwann cells.

Peripheral neuropathy is a serious diabetic complication. Delayed nerve regeneration in diabetic animal models suggests abnormalities in proliferation/differentiation of Schwann cells (SC). We recently reported that endothelins (ETs) regulate proliferation and phenotype in primary and immortalized SC (iSC). We now investigated changes in the effects of ETs on SC proliferation and signaling in nerve segments from streptozotocin-induced diabetic rats and in iSC exposed to high glucose. Cultured explants from diabetic rats displayed a delay in the time-course of [3H]-thymidine incorporation as well as enhanced sensitivity to endothelin-1 (ET-1) or insulin. iSC cultured in high (25 mM) glucose-containing media also exhibited higher [3H]-thymidine incorporation, along with an enhanced activation of p38 mitogen-activated protein kinase and phospholipase C in response to ET-1 or platelet-derived growth factor as compared to controls (5.5 mM glucose). These studies support an extra-vascular role of ETs in peripheral nerves and SC. The increased sensitivity to ET-1 in nerves and iSC exposed to high glucose may contribute to abnormal SC proliferation characterizing diabetic neuropathy.