tetrodotoxin and Diabetes-Mellitus--Type-1

Intracellular Na(+) ([Na(+)](i)) is an important modulator of excitation-contraction coupling via regulating Ca(2+) efflux/influx, and no investigation has been so far performed in diabetic rat heart. Here, we examined whether any change of [Na(+)](i) in paced cardiomyocytes could contribute to functional alterations during diabetes. Slowing down in depolarization phase of the action potential, small but significant decrease in its amplitude with a slight depolarized resting membrane potential was traced in live cardiomyocytes from diabetic rat, being parallel with a decreased TTX-sensitive Na(+) channel current (I(Na)) density. We recorded either [Na(+)](i) or [Ca(2+)](i) by using a fluorescent Na(+) indicator (SBFI-AM or Na-Green) or a Ca(2+) indicator (Fura 2-AM) in freshly isolated cardiomyocytes. We examined both [Na(+)](i) and [Ca(2+)](i) at rest, and also [Na(+)](i) during pacing with electrical field stimulation in a range of 0.2-2.0 Hz stimulation frequency. In order to test the possible contribution of Na(+)/H(+) exchanger (NHE) to [Na(+)](i), we examined the free cytoplasmic [H(+)](i) changes from time course of [H(+)](i) recovery in cardiomyocytes loaded with SNARF1-AM by using ammonium prepulse method. Our data showed that [Na(+)](i) in resting cells from either diabetic or control group was not significantly different, whereas the increase in [Na(+)](i) was significantly smaller in paced diabetic cardiomyocytes compared to that of the controls. However, resting [Ca(2+)](i) in diabetic cardiomyocytes was significantly higher than that of the controls. Here, a lower basal pH(i) in diabetics compared with the controls correlates also with a slightly higher but not significantly different NHE activity and consequently a similar Na(+) loading rate at resting state with a leftward shift in pH sensitivity of NHE-dependent H(+)-flux. NHE protein level remained unchanged, while protein levels of Na(+)/K(+) ATPase and Na(+)/Ca(2+) exchanger were decreased in the diabetic cardiomyocytes. Taken together, the present data indicate that depressed I(Na) plays an important role in altered electrical activity with less Na(+) influx during contraction, and an increased [Ca(2+)](i) load in these cells seems to be independent of [Na(+)](i). The data with insulin treatment suggest further a recent balance between Na(+) influx and efflux proteins associated with the [Na(+)](i), particularly during diabetes.

Topics: Action Potentials; Animals; Cytoplasm; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Heart Ventricles; Hydrogen-Ion Concentration; Insulin; Male; Myocytes, Cardiac; Papillary Muscles; Patch-Clamp Techniques; Rats; Rats, Wistar; Sodium; Tetrodotoxin; Voltage-Gated Sodium Channels

Bladder dysfunction is a common complication of diabetic autonomic neuropathy; however, its cause remains uncertain. We have recently identified a novel IgG autoantibody (Ab) in patients with type 1 diabetes that acts as an agonist at the dihydropyridine (DHP) site of L-type voltage-gated calcium channels (VGCC), disrupting neuronal regulation of visceral smooth muscle. In the present study, passive transfer to mice of IgG from patients with type 1 diabetes was used to investigate the role of anti-VGCC Abs in mediating diabetic bladder dysfunction. Injection of mice with diabetic immunoglobulin (IgG) with anti-VGCC activity induced features of an overactive bladder, including phasic detrusor contractions and a loss of bladder wall compliance. The bladder overactivity is mimicked by the DHP agonist Bay K8644, reversed by the DHP antagonist nicardipine, but is insensitive to the motor nerve blocker tetrodotoxin, indicating that the anti-VGCC Ab acts at the level of the bladder detrusor itself. This study reports the first evidence of Ab-mediated bladder dysfunction in type 1 diabetes, which may be part of a wider spectrum of smooth muscle and cardiac abnormalities.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Autoantibodies; Calcium Channels, L-Type; Diabetes Mellitus, Type 1; Male; Mice; Mice, Inbred BALB C; Nicardipine; Tetrodotoxin; Urinary Bladder; Urinary Bladder Diseases