guanosine-diphosphate and Diabetic-Neuropathies

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.

Topics: Analgesics, Opioid; Animals; Biological Transport; Calcium; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Neuropathies; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Ganglia, Spinal; GTP Phosphohydrolases; GTP-Binding Protein alpha Subunits, Gi-Go; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; In Vitro Techniques; Male; Neural Conduction; Neurons; Patch-Clamp Techniques; Pertussis Toxin; Rats; Rats, Inbred BB; Rats, Sprague-Dawley; Receptors, Opioid, mu; Sulfur Radioisotopes; Thionucleotides; Virulence Factors, Bordetella

The regulation of phosphoinositidase C (PIC) activity by guanosine-5'-(3-O-thio)triphosphate (GTP gamma S) was characterized in a cholate-solubilized peripheral myelin-enriched fraction from rat sciatic nerve. The GTP analog maximally enhanced PIC-catalyzed hydrolysis of exogenous phosphatidylinositol-4,5-bisphosphate (PIP2) in a dose-dependent manner only within a narrow range of cholate concentrations. Maximal stimulation was attained at 0.6 microM GTP gamma S and could be completely prevented by 1 microM guanosine-5'-(2-O-thio)diphosphate. Neither adenylyl-imidodiphosphate nor adenosine triphosphate (ATP) enhanced PIC activity. Carbamoylcholine (1 mM) added together with GTP gamma S increased the extent of PIP2 hydrolysis over that elicited by GTP gamma S alone and this stimulation was blocked by the muscarinic receptor antagonist, atropine (50 microM). In detergent-solubilized myelin preparations from streptozotocin-induced diabetic rats, a higher concentration of the guanine nucleotide analog was required to achieve stimulation comparable to that obtained with corresponding preparations from normal animals. These results suggest that sciatic nerve myelin possesses muscarinic receptors coupled via a GTP-binding protein to PIC and that this system can be reconstituted in detergent-solubilized extracts. It is possible that the function of G proteins in cell signaling is impaired in experimental diabetic neuropathy.

Topics: Adenosine Triphosphate; Adenylyl Imidodiphosphate; Animals; Atropine; Carbachol; Cholic Acid; Cholic Acids; Diabetes Mellitus, Experimental; Diabetic Neuropathies; Enzyme Activation; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Male; Myelin Sheath; Peripheral Nerves; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phosphoric Diester Hydrolases; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Sciatic Nerve; Streptozocin; Thionucleotides