Xiaoling Dai

Increased O2·- Production and Upregulation of ETB Receptors by Sympathetic Neurons in DOCA-Salt Hypertensive Rats

Abstract—Superoxide anion (O2·−) production is elevated in the vasculature of hypertensive animals but it is not known if O2·− production is also elevated in the sympathetic nervous system. We measured O2·− levels in prevertebral sympathetic ganglia of deoxycorticosterone acetate (DOCA)-salt hypertensive rats using the dihydroethidine (DHE) fluorescence method. O2·− was elevated in ganglia from DOCA-salt rats compared with normotensive sham rats. Treatment of ganglia with endothelin (ET)-1 (3×10−8 mol/L) resulted in a 200% increase in fluorescence intensity in neurons, which was attenuated by the ETB receptor antagonist BQ788 (10−7 mol/L). ET-1 also increased the O2·− induced fluorescence in dissociated sympathetic neurons and PC-12 cells via activation of ETB receptors, but not ETA receptors. To evaluate whether elevated ET-1 levels in the ganglia might contribute to the elevated O2·− found in ganglia we measured the amount of ET-1 using an ELISA assay. ET-1 levels in sham rat celiac ganglia were 695.6±40.9 picogram per gram; they were not different than ET-1 levels in ganglia from DOCA-salt rats. We then compared ETB receptor levels in ganglia from sham and DOCA-salt animals. ETB receptor mRNA levels were 32% higher and ETB receptor protein levels were 20% higher in celiac ganglia from DOCA-salt rats than from sham rats separately. In conclusion, O2·− is elevated in prevertebral sympathetic ganglia in DOCA-salt hypertension, and ET-1 is a potent stimulus for the elevation of O2·− levels in sympathetic ganglia, an effect that may be mediated by the upregulation of ETB receptors.

Differential trafficking and desensitization of human ET(A) and ET(B) receptors expressed in HEK 293 cells.

Endothelin-1 (ET-1) is a vasoconstrictor peptide that acts on ETA and ETB receptors on smooth muscle cells (SMCs). Because vascular SMCs can express both receptors, it is difficult to study the localization and properties of each subtype. Therefore, we investigated the localization and function of ETA and ETB receptors transfected into HEK 293 cells. Immunocytochemistry was used to examine colocalization of ET receptors with the plasma membrane marker, pan cadherin. In cells transfected with ETA receptors, 83 ± 2% of these receptors colocalized with pan cadherin. In ETB receptor–transfected cells, 54 ± 2% of the receptor colocalized with pan cadherin. When ETA and ETB receptors were cotransfected, 97 ± 1% of ETB receptors colocalized with ETA receptors and 84 ± 2% of ETB receptors colocalized with pan cadherin. ET-1 and sarafotoxin 6c (S6c, ETB receptor agonist) increased [Ca2+]i in cells transfected with ETA or ETB receptors; 100 nM of ET-1 and S6c caused maximal responses. When stimulated with ET-1, ETB receptors desensitized faster (t1/2 = 21 ± 1 sec) than ETA receptors (t1/2 = 48 ± 1 sec). S6c-induced increases in [Ca2+]i desensitized in cells expressing ETB receptors only (t1/2 = 17 ± 1 s). Desensitization was eliminated in cells cotransfected with ET receptors. We conclude that ETA receptors localize to the cell membrane, whereas ETB receptors are in the membrane and intracellular compartments. Coexpressed ET receptors are in the membrane. ETB receptors desensitize faster than ETA receptors, but receptor coexpression eliminates desensitization. Finally, ETA and ETB receptors interact to change receptor trafficking which may modify ET receptor function in vascular SMCs coexpressing these receptors.

Differential Regulation of NADPH Oxidase in Sympathetic and Sensory Ganglia in Deoxycorticosterone Acetate–Salt Hypertension

We demonstrated recently that superoxide anion levels are elevated in prevertebral sympathetic ganglia of deoxycorticosterone acetate–salt hypertensive rats and that this superoxide anion is generated by reduced nicotinamide-adenine dinucleotide phosphate oxidase. In this study we compared the reduced nicotinamide-adenine dinucleotide phosphate oxidase enzyme system of dorsal root ganglion (DRG) and sympathetic celiac ganglion (CG) and its regulation in hypertension. The reduced nicotinamide-adenine dinucleotide phosphate oxidase activity of ganglion extracts was measured using fluorescence spectrometry of dihydroethidine; the activity in hypertensive dorsal root ganglion was 34% lower than in normotensive DRG. In contrast, activity was 79% higher in hypertensive CG than normotensive CG. mRNA for the oxidase subunits NOX1, NOX2, NOX4, p47phox, and p22phox were present in both CG and DRG; mRNA for NOX4 was significantly higher in CG than in DRG. The levels of mRNA and protein expression of the membrane-bound catalytic subunit p22phox and of the regulatory subunits p47phox and Rac-1 were measured in CG and DRG in normotensive and hypertensive rats. p22phox mRNA and protein expression was greater in CG of hypertensive rats but not in DRG. Compared with normotensive controls, p47phox mRNA and protein, as well as Rac-1 protein, were significantly decreased in hypertensive DRG but not in CG. Immunohistochemical staining of p47phox showed translocation from cytoplasm to membrane in hypertensive CG but not in hypertensive DRG. This suggests that reduced nicotinamide-adenine dinucleotide phosphate oxidase activation in sympathetic neurons and sensory neurons is regulated in opposite directions in hypertension. This differential regulation may contribute to unbalanced vasomotor control and enhanced vasoconstriction in the splanchnic circulation.

Inhibitory neuromuscular transmission to ileal longitudinal muscle predominates in neonatal guinea pigs

Background  Inhibitory neurotransmission to the longitudinal muscle is more prominent in the neonatal than in the adult guinea pig ileum.

R‐type Ca2+ channels contribute to fast synaptic excitation and action potentials in subsets of myenteric neurons in the guinea pig intestine

Background  R‐type Ca2+ channels are expressed by myenteric neurons in the guinea pig ileum but the specific function of these channels is unknown.

Increased superoxide levels in ganglia and sympathoexcitation are involved in sarafotoxin 6c-induced hypertension.

Endothelin (ET) type B receptors (ET(B)R) are expressed in multiple tissues and perform different functions depending on their location. ET(B)R mediate endothelium-dependent vasodilation, clearance of circulating ET, and diuretic effects; all of these should produce a fall in arterial blood pressure. However, we recently showed that chronic activation of ET(B)R in rats with the selective agonist sarafotoxin 6c (S6c) causes sustained hypertension. We have proposed that one mechanism of this effect is constriction of capacitance vessels. The current study was performed to determine whether S6c hypertension is caused by increased generation of reactive oxygen species (ROS) and/or activation of the sympathetic nervous system. The model used was continuous 5-day infusion of S6c into male Sprague-Dawley rats. No changes in superoxide anion levels in arteries and veins were found in hypertensive S6c-treated rats. However, superoxide levels were increased in sympathetic ganglia from S6c-treated rats. In addition, superoxide levels in ganglia increased progressively the longer the animals received S6c. Treatment with the antioxidant tempol impaired S6c-induced hypertension and decreased superoxide levels in ganglia. Acute ganglion blockade lowered blood pressure more in S6c-treated rats than in vehicle-treated rats. Although plasma norepinephrine levels were not increased in S6c hypertension, surgical ablation of the celiac ganglion plexus, which provides most of the sympathetic innervation to the splanchnic organs, significantly attenuated hypertension development. The results suggest that S6c-induced hypertension is partially mediated by sympathoexcitation to the splanchnic organs driven by increased oxidative stress in prevertebral sympathetic ganglia.