Protection against lipopolysaccharide-induced endothelial dysfunction in resistance and conduit vasculature of iNOS knockout mice.

Endothelial dysfunction is a characteristic of, and may be pathogenic in, inflammatory cardiovascular diseases, including sepsis. The mechanism underlying inflammation-induced endothelial dysfunction may be related to the expression and activity of inducible nitric oxide synthase (iNOS). This possibility was investigated in isolated resistance (mesenteric) and conduit (aorta) arteries taken from lipopolysaccharide (LPS)-treated (12.5 mg/kg i.v.) or saline-treated iNOS knockout (KO) and wild-type (WT) mice. LPS pretreatment (for 15 h, but not 4 h) profoundly suppressed responses to acetylcholine (ACh) and significantly reduced sensitivity to the NO donor spermine-NONOate (SPER-NO) in aorta and mesenteric arteries of WT mice. This effect was temporally associated with iNOS protein expression in both conduit and resistance arteries and with a 10-fold increase in plasma NOx levels. In contrast, no elevation of plasma NOx was observed in LPS-treated iNOS KO animals, and arteries dissected from these animals did not express iNOS or display hyporeactivity to ACh or SPER-NO. The mechanism underlying this phenomenon may be suppression of eNOS expression, as observed in arteries of WT animals, that was absent in arteries of iNOS KO animals. These results clearly demonstrate that iNOS induction plays an integral role in mediation of the endothelial dysfunction associated with sepsis in both resistance and conduit arteries.

Effects of nerve growth factor on expression of GAP-43 in right atria after sympathectomy in diabetic rats.

AIM: The present study investigated the role of nerve growth factor (NGF) in the regeneration of noradrenergic nerves of right atria (following 6-hydroxydopamine; 6-OHDA, 100 mg/kg, i.p.) from non-diabetic and 8-week diabetic rats. RESULTS: In cryostat sections of the right atria, GAP-43 immunoreactivity was concentrated in nerve terminals, preterminal axons of the endocardium, epicardium and myocardium, as well as in nerve fibres innervating the blood vessels and ganglionic cells. In serial sections, all positive staining for GAP-43 showed immunoreactivity for the neuronal marker PGP-9.5. In untreated non-diabetic rats, the total GAP-43 immunoreactivity was reduced to 60% relative to pretreatment levels, at day 14 after 6-OHDA, as quantified by Western blotting. In diabetic rats, 6-OHDA treatment produced a marked increase in the levels of total GAP-43 at days 28 and 49. NGF treatment (1 mg/kg, s.c., 3 times/week, for 2 weeks) had no effect on the level of total GAP-43 in right atria from non-diabetic and diabetic rats before treatment with 6-OHDA. However, it normalized the reduced GAP-43 immunoreactivity observed in 6-OHDA-treated non-diabetic rats. Interestingly, NGF treatment alone produced an increase in GAP-43 phosphorylation relative to total GAP-43 in right atria from both non-diabetic (44%) and diabetic groups (42%). CONCLUSIONS: These findings suggest that nerve terminals of the right atria retain, in the mature adult, the capacity for structural and functional plasticity. The expression of GAP-43 in right atria of control and diabetic rats was differentially affected by 6-OHDA treatment. In injured noradrenergic neurones of the right atria, NGF modified the expression of GAP-43 only in non-diabetic rats and not in diabetic rats.

The regeneration of peripheral noradrenergic nerves after chemical sympathectomy in diabetic rats: effects of nerve growth factor.

The study investigated the role of nerve growth factor (NGF) in the regeneration of noradrenergic nerves of the right atria from control and 8-week diabetic rats, after lesion caused by a single injection of 6-hydroxydopamine (6-OHDA, 100 mg/kg ip). This treatment caused a profound depletion of tissue noradrenaline (NA) of the right atria from both control and diabetic groups, followed by a progressive repletion that was not complete at 49 days. Immunoreactivity for the NGF receptors trkA and p75(NTR) was decreased and increased, respectively, between days 3 and 28 in right atria from diabetic rats and returned to pretreatment levels at day 49. Receptor levels were not significantly altered in controls. In contrast to tissue NA, at day 14 functional responses to electrical nerve stimulation of the right atria had completely returned to the pretreatment state in diabetic rats and were very close to normal in nondiabetic rats. NGF treatment (1 mg/kg, three times/week, for 2 weeks) increased tissue NA only in control rats; the pattern was similar after 6-OHDA. These findings are consistent with the hypothesis that NGF normally plays a role in the regulation of autonomic sympathetic nerves in the adult rat atrium and that mature and uninjured sympathetic neurons remain responsive to NGF. In injured noradrenergic neurons, NGF promotes regeneration in nondiabetic rats. The ability of NGF to promote regeneration of noradrenergic nerves is lost in diabetes and this may relate to the loss of trkA receptor on prejunctional nerve terminals after denervation.

Reduced nerve blood flow in diabetic rats: relationship to nitric oxide production and inhibition of aldose reductase.

This study examined links between impaired nitric oxide production in the sciatic endoneurium, nerve blood flow, and polyol pathway flux, to test the hypothesis that reduced nerve blood flow might be compromised by competition for NADPH between aldose reductase and nitric oxide synthase. Sciatic nerves of streptozotocin-diabetic rats showed reduced laser Doppler flux (by 51% or 63%; both p<0.05)-indicative of reduced nerve blood flow-and reduced motor nerve conduction velocity (17% in two experiments; p<0.05). Acute interruption of nitric oxide production in the sciatic nerves of control rats, via endoneurial injection of N omega-nitro-D-arginine methyl ester (L-NAME), caused a local reduction (of 64%; p<0.001) in nerve Doppler flux. This was reversed by either L-arginine or sodium nitroprusside. The response to L-NAME was greatly reduced in diabetic rats (only 22% reduction; p<0.01), though both L-arginine and SNP caused marked increases in flux. Chronic inhibition of aldose reductase in diabetic rats (with either sorbinil or imirestat at a range of doses) had little effect on resting sciatic nerve Doppler flux, though both inhibitors normalized conduction velocity. Both aldose reductase inhibitors reduced sorbitol pathway intermediates in a dose-related manner. These findings do not support the proposition that aldose reductase inhibitors normalise conduction velocity by mechanisms dependent upon either normalization of endoneurial nitric oxide or nerve blood flow. Instead, a mechanism based upon more direct effects on axon or Schwann cell function is favoured.

Effects of adrenergic stimulation on sciatic nerve blood flow in diabetic and control rats.

Sciatic endoneurial blood flow is reduced in experimental diabetes. This study examined the possible involvement of noradrenergic mechanisms in this impairment. In anaesthetised rats (pentobarbitone sodium 50 mg/kg, diazepam 2 mg/kg), sciatic nerve laser Doppler flux and vascular resistance in diabetic rats (5-6 weeks) were lower (approximately 50%) and higher (approximately 42%), respectively, than that in age-matched control rats, indicating nerve ischaemia in the diabetic tissues. Tyramine (1 nmol), noradrenaline (0.001-1 nmol) and phenylephrine (0.01-10 nmol) produced significant increases of nerve vascular resistance in control rats. The responses to tyramine (1 nmol) were completely blocked by desipramine (10 nmol) and those to phenylephrine (10 nmol) were reversed by phentolamine (1 nmol). In streptozotocin-diabetic rats, responses to phenylephrine or noradrenaline were enhanced compared to control rats, but the enhancement failed to reach statistical significance. The findings demonstrate that adrenergic stimulation affects sciatic nerve endoneurial blood flow.

Attenuation of vasoconstriction by endogenous nitric oxide in rat caudal artery.

1. The effects of NG-nitro-L-arginine (L-NNA), NG-nitro-L-arginine methyl ester (L-NAME), haemoglobin and methylene blue have been examined on vascular reactivity in the rat isolated caudal artery. The effects of L-NNA and sodium nitroprusside were also investigated on the stimulation-induced (S-I) efflux of noradrenaline in the rat caudal artery. 2. L-NNA (10 microM) and L-NAME (10 microM) significantly attenuated the vasodilator responses to acetylcholine (1 nM-1 microM), but had no effect on vasodilator responses to papaverine (1-100 microM). 3. Vasoconstrictor responses to sympathetic nerve stimulation (3 Hz, 10 s), noradrenaline (0.01-1 microM), methoxamine (1-10 microM), 5-hydroxytryptamine (0.01-0.3 microM), phenylephrine (0.1-10 microM), endothelin-1 (10 nM) and KCl (40 mM) were significantly enhanced by 10 microM L-NNA. L-NAME (10 microM) caused a significant enhancement of vasoconstrictor responses to noradrenaline and sympathetic nerve stimulation in endothelium-intact, but not in endothelium-denuded tissues. 4. Haemoglobin and methylene blue (both 10 microM) enhanced the vasoconstrictor responses to sympathetic nerve stimulation and noradrenaline. The enhancements were absent in endothelium-denuded arterial segments. 5. In endothelium-denuded arterial segments precontracted with phenylephrine, the vasodilator responses to the nitric oxide donor, sodium nitroprusside (0.1-300 nM) were decreased by increasing the level of precontraction. 6. L-NNA (10 microM) had no effect on the S-I efflux of radioactivity from arteries in which transmitter stores had been labelled with [3H]-noradrenaline. 7. These results suggest that endothelial nitric oxide attenuates vasoconstrictor responses in the rat caudal artery through activation of soluble guanylate cyclase to decrease smooth muscle contractility. Therefore, the findings provide evidence that nitric oxide acts as a functional antagonist to oppose vasoconstriction.

Endothelial nitric oxide attenuates vasoconstrictor responses to nerve stimulation and noradrenaline in the rat tail artery.

The effects of the nitric oxide synthesis inhibitor, NG-nitro-L-arginine (NOLA), have been examined in perfused segments of rat tail artery. NOLA (1 and 10 microM) significantly enhanced the vasoconstrictor responses to perivascular nerve stimulation (5 Hz, 10 s) and noradrenaline (10 ng). The enhancing effects of NOLA were prevented by L-arginine, but not by D-arginine, and were absent in endothelium-denuded artery segments. The results suggest that nitric oxide derived from endothelial cells attenuates vasoconstrictor responses to both nerve stimulation and noradrenaline.

Modulation of norepinephrine-induced vasoconstriction by endothelin-1 and nitric oxide in rat tail artery.

The effects of endothelin-1 (ET-1) and an inhibitor of the synthesis of nitric oxide (NO) have been examined on vasoconstrictor responses to nerve stimulation and norepinephrine in the rat isolated perfused tail artery. In endothelium-denuded preparations, a 60-min exposure to 0.3 nM ET-1 had no effect on the basal perfusion pressure, but significantly enhanced responses to stimulation (1 Hz, 10 s) and norepinephrine (10 ng) to 124 +/- 9% (n = 6) and 139 +/- 14% (n = 8), respectively, of control responses. In endothelium-intact preparations, inhibition of NO synthesis by NG-nitro-L-arginine (NOLA, 10 microM) enhanced responses to stimulation (5 Hz, 10 s) and norepinephrine (10 ng) to 171 +/- 12% (n = 6) and 222 +/- 9% (n = 4), respectively, of control responses. The NOLA-induced enhancements were prevented by 100 microM L-arginine but not by 100 microM D-arginine, and did not occur in endothelium-denuded arteries. In other experiments, segments of rat tail artery were perfused in a low-volume (3.5 ml) recirculating system. The basal perfusion pressure was consistent for at least 60 min. In endothelium-denuded segments, vasoconstrictor responses to nerve stimulation (0.5 Hz, 10 s) or norepinephrine (10 ng) remained constant. However, in endothelium-intact segments, responses to stimulation and norepinephrine gradually increased to 158 +/- 13% (n = 6) and 152 +/- 8% (n = 5), respectively, of initial responses, after 45 min of recirculation. The increases were not related to NO, as they remained unchanged in the presence of 10 microM NOLA. Thus, it appears that a vasoconstriction-enhancing factor, possibly ET-1, is released from endothelial cells and accumulates in the perfusion fluid.