Zhongling Zheng

Vagal afferent control of opioidergic effects in rat brainstem circuits

We demonstrated recently that increasing the levels of cAMP allows opioids to modulate GABAergic synaptic transmission between the nucleus of the tractus solitarius (NTS) and dorsal motor nucleus of the vagus (DMV). Using a combination of electrophysiological, immunohistochemical and biochemical approaches, we provide evidence that vagal afferent fibres dampen cAMP levels within the vagal brainstem circuits via tonic activation of group II metabotropic glutamate receptors (mGluRs). Whole‐cell patch‐clamp recordings were made from identified neurons of the rat DMV. Following chronic vagal deafferentation, the opioid agonist methionine‐enkephalin (ME) inhibited the amplitude of evoked IPSC (eIPSC) in 32 of 33 neurons, without exogenous enhancement of cAMP levels. The ME‐induced inhibition was prevented by the group II mGluR‐selective agonist APDC. Following perfusion with the group II mGluR‐selective antagonist EGLU, ME inhibited eIPSC amplitude in brainstem slices of control rats. Immunohistochemical experiments revealed that, following vagal deafferentation, μ‐opioid receptors were colocalized on GABAergic profiles apposing DMV neurons; the number of colocalized profiles was significantly decreased by pretreatment with APDC. Radioimmunoassay and Western blot analysis showed that cAMP and phosphorylated cyclic AMP response element binding protein (pCREB) levels in the dorsal vagal complex were increased following vagal deafferentation. Our data show that by tonically dampening the levels of cAMP within the GABAergic synaptic contacts, activated group II mGluRs prevent the modulation of this synapse by endogenous opioids. These data suggest that the plasticity, hence the response, of central circuits controlling the vagal motor outflow to visceral organs is modulated and finely tuned by vagal afferent fibres.

Nitric oxide is a sensory nerve neurotransmitter in the mesenteric artery of guinea pig

Previous studies have shown that the guinea pig inferior mesenteric artery receives spinal sensory vasodilatory innervation, which can be activated by colon distention and electrical nerve stimulation. In the present study, we investigated the hypotheses that nitric oxide synthase (NOS) is present in guinea pig primary sensory neurons in the dorsal root ganglion (DRG) and in nerve fibers surrounding the mesenteric arteries, and that nitric oxide (NO) is a sensory neurotransmitter in the inferior mesenteric artery in vitro. Double-labeling immunohistochemistry showed that neuronal NOS-IR was found in 12% of cells of guinea pig thoracic and lumbar DRGs; in 95.1% of these cells it was colocalized with substance P (SP), and SP immunoreactivity (SP-IR) was present in 23% of cells of the same DRGs. Neuronal NOS-like immunoreactivity was localized in nerve fibers surrounding guinea pig mesenteric artery and 25% of them were double stained with SP-IR. Endothelium-denuded inferior mesenteric artery preparations in vitro were incubated with guanethidine (30 microns, 30 min) and pre-contracted with methoxamine (30 microns). The NO donors, sodium nitroprusside (1 micron) and L-nitrosocysteine (300 microns), produced 91.0 +/- 5.5 and 90.4 +/- 9.6% vasodilatation of total vasodilatation in the vessel segments, respectively, which was capsaicin- or tetrodotoxin-insensitive. Repetitive electrical field stimulation of the preparations produced a frequency-dependent vasodilatation which was reduced by pretreatment with capsaicin or by tetrodotoxin (10 microns). The NOS inhibitor N omega-nitro-L-arginine (L-NNA) (100 microns, 30 min) diminished the nerve-evoked vasodilatation from 41.8 +/- 8.4 to 21.4 +/- 9.7% at 2 Hz and from 50.8 +/- 5.6 to 19.0 +/- 7.3% at 15 Hz (P < 0.05), whereas NG-nitro-L-arginine methyl ester (L-NAME, 100 microns-1 mM) did not significantly inhibit the relaxation. The stereo isomer nitro-D-arginine (D-NNA, 100 microns, 30 min) was ineffective. These findings suggest that NO is a neurotransmitter released from primary sensory nerves which mediates vasodilation in vitro.

Two populations of sympathetic neurons project selectively to mesenteric artery or vein

The objective of this study was to determine whether sympathetic neurons of the inferior mesenteric ganglion (IMG) projecting to mesenteric arteries could be distinguished by their localization, neurochemical phenotype, and electrophysiological properties from neurons projecting to mesenteric veins. In an in vitro intact vasculature-IMG preparation, neurons were labeled following intraluminal injection of Fluoro-Gold or rhodamine beads into the inferior mesenteric artery (IMA) or vein (IMV). The somata of neurons projecting to IMA were localized in the central part of the IMG, whereas those projecting to IMV were localized more peripherally. None of the labeled neurons was doubly labeled. Neuropeptide Y immunoreactivity was found in 18.9% of neurons innervating the IMA, but not in neurons innervating the IMV. Identified neurons were dissociated and characterized using whole cell patch-clamp recording. After direct soma depolarization, all of the labeled arterial and venous neurons were classified as tonic firing, compared with only 40% of unlabeled neurons; the remaining 60% of unlabeled neurons were phasic firing. The results indicate that IMG neurons projecting to mesenteric arteries are distinct from neurons projecting to mesenteric veins.The objective of this study was to determine whether sympathetic neurons of the inferior mesenteric ganglion (IMG) projecting to mesenteric arteries could be distinguished by their localization, neurochemical phenotype, and electrophysiological properties from neurons projecting to mesenteric veins. In an in vitro intact vasculature-IMG preparation, neurons were labeled following intraluminal injection of Fluoro-Gold or rhodamine beads into the inferior mesenteric artery (IMA) or vein (IMV). The somata of neurons projecting to IMA were localized in the central part of the IMG, whereas those projecting to IMV were localized more peripherally. None of the labeled neurons was doubly labeled. Neuropeptide Y immunoreactivity was found in 18.9% of neurons innervating the IMA, but not in neurons innervating the IMV. Identified neurons were dissociated and characterized using whole cell patch-clamp recording. After direct soma depolarization, all of the labeled arterial and venous neurons were classified as tonic firing, compared with only 40% of unlabeled neurons; the remaining 60% of unlabeled neurons were phasic firing. The results indicate that IMG neurons projecting to mesenteric arteries are distinct from neurons projecting to mesenteric veins.

N-hydroxyl derivatives of guanidine based drugs as enzymatic NO donors.

Recent research suggests that NO may play a role in the physiological effects of some guanidine-containing drugs. In this report, three guanidine-containing drugs (guanadrel, guanoxan, and guanethidine) together with their N-hydroxyl derivatives were synthesized and their NO-releasing abilities catalyzed by nitric oxide synthases (NOSs) and horseradish peroxidase were evaluated. The guanidine containing compounds could not release NO in the presence of NOS or peroxidase. The corresponding N-hydroxyl compounds exhibited weak NO-releasing ability under the catalyzed of NOS and good NO-releasing ability under the oxidation by horseradish peroxidase in the presence of H(2)O(2). These compounds also displayed vasodilatory activity.

Guanethidine evokes vasodilatation in guinea pig mesenteric artery by acting on sensory nerves

In precontracted, endothelium-free guinea pig mesenteric artery rings, in which adrenergic vasoconstrictor responses had been eliminated, guanethidine (1-30 microM) produced a vasodilatation of 69.3+/-4.4%. The vasodilatation was reduced 89% by capsaicin (10 microM) or 55% by tetrodotoxin (10 microM), indicating mediation of this effect by primary sensory nerves. The nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (100 microM, 30 min) but not its stereoisomer reduced the guanethidine vasodilatation by 70%. Blockade of monoamine uptake with ouabain (25 microM, 15 min) or cocaine (5 microM, 5 min) reduced the guanethidine-induced vasodilatation by 85 and 67%, respectively. These results suggest that guanethidine produced vasodilatation by being transported into capsaicin-sensitive primary sensory nerves where it functioned as a substrate for nitric oxide synthase to generate a vasodilatory substance.