Nitric oxide control of lower vertebrate blood vessels by vasomotor nerves

In mammals, much is understood about the endothelial and neural NO control mechanisms in the vasculature. In contrast, NO control of blood vessels in lower vertebrates is poorly understood, with the majority of research focusing on the presence of an endothelial NO system; however, its presence remains controversial. This study examined the mechanisms by which NO regulates the large blood vessels of non-mammalian vertebrates. In all species examined, the arteries and veins contained a plexus of NOS-positive perivascular nerves that included nerve bundles and fine, varicose nerve terminals. However, in the large arteries and veins of various species of fishes and amphibians, no anatomical evidence was found for endothelial NOS using both NADPH-diaphorase and eNOS immunohistochemistry. In contrast, perinuclear NOS staining was readily apparent in blue-tongue lizard, pigeon and rat, which suggested that eNOS first appeared in reptiles. Physiological analysis of NO signalling in the vascular smooth muscle of short-finned eel and cane toad could not find any evidence for endothelial NO signalling. In contrast, it appears that activation of the nitrergic vasomotor nerves is responsible for NO control of the blood vessels.

Chemical coding of vascular nitrergic nerves in lower vertebrates

Nitric oxide is an important regulator of blood pressure in mammals. This study provided new information on the role of nitric-oxide releasing sympathetic nerves in vascular regulation of lower vertebrates. The research outcomes advance knowledge on the potential role of these unique nerves in the control of the lower vertebrate cardiovascular system.

Altered responsiveness of the kidney to activation of the renal nerves in fat-fed rabbits

We tested whether mild adiposity alters responsiveness of the kidney to activation of the renal sympathetic nerves. After rabbits were fed a high-fat or control diet for 9 wk, responses to reflex activation of renal sympathetic nerve activity (RSNA) with hypoxia and electrical stimulation of the renal nerves (RNS) were examined under pentobarbital anesthesia. Fat pad mass and body weight were, respectively, 74% and 6% greater in fat-fed rabbits than controls. RNS produced frequency-dependent reductions in renal blood flow, cortical and medullary perfusion, glomerular filtration rate, urine flow, and sodium excretion and increased renal plasma renin activity (PRA) overflow. Responses of sodium excretion and medullary perfusion were significantly enhanced by fat feeding. For example, 1 Hz RNS reduced sodium excretion by 79 ± 4% in fat-fed rabbits and 46 ± 13% in controls. RNS (2 Hz) reduced medullary perfusion by 38 ± 11% in fat-fed rabbits and 9 ± 4% in controls. Hypoxia doubled RSNA, increased renal PRA overflow and medullary perfusion, and reduced urine flow and sodium excretion, without significantly altering mean arterial pressure (MAP) or cortical perfusion. These effects were indistinguishable in fat-fed and control rabbits. Neither MAP nor PRA were significantly greater in conscious fat-fed than control rabbits. These observations suggest that mild excess adiposity can augment the antinatriuretic response to renal nerve activation by RNS, possibly through altered neural control of medullary perfusion. Thus, sodium retention in obesity might be driven not only by increased RSNA, but also by increased responsiveness of the kidney to RSNA.

Nitric oxide regulation of the central aortae of the toad Bufo marinus occurs independently of the endothelium

Nitric oxide (NO) signalling pathways were examined in the lateral aortae and dorsal aorta of the cane toad Bufo marinus. NADPH diaphorase histochemistry and nitric oxide synthase (NOS) immunohistochemistry found no evidence for endothelial NOS in the endothelium of toad aortae, but it could be readily demonstrated in rat aorta that was used as a control. Immunohistochemistry using a specific neural NOS antibody showed the presence of neural NOS immunoreactivity in the perivascular nerves of the aortae. The anatomical data was supported by in vitro organ bath physiology, which demonstrated that the vasodilation mediated by applied acetylcholine (10^-5 mol l^-1) was not dependent on the presence of the vascular endothelium; however, it was significantly reduced in the presence of a neural NOS inhibitor, vinyl-L-NIO (10-4 mol l^-1). In addition, atropine (10^-6 mol l^-1) (a muscarinic receptor inhibitor), L-NNA (10^-4 mol l^-1) (a NOS inhibitor) and ODQ (10^-5 mol l^-1) (an inhibitor of soluble guanylyl cyclase) abolished the vasodilatory effect of applied acetylcholine. In conclusion, we propose that an endothelial NO system is absent in toad aortae and that NO generated by neural NOS in perivascular nerves mediates vasodilation.

Nitric oxide control of the dorsal aorta and the intestinal vein of the Australian short-finned eel Anguilla australis

This study investigated the mechanisms by which nitric oxide (NO) regulates the dorsal aorta and the intestinal vein of the Australian short-finned eel Anguilla australis. NADPH diaphorase histochemistry and immunohistochemistry using a mammalian endothelial nitric oxide synthase (NOS) antibody could not demonstrate NOS in the endothelium of either blood vessel; however, NOS could be readily demonstrated in the endothelium of the rat aorta that was used as a control. Both blood vessels contained NADPH diaphorase positive nerve fibres and nerve bundles, and immunohistochemistry using a neural NOS antibody showed a similar distribution of neural NOS immunoreactivity in the perivascular nerves. In vitro organ bath physiology showed that a NO/soluble guanylyl cyclase (GC) system is present in the dorsal aorta and the intestinal vein, since the soluble GC inhibitor oxadiazole quinoxalin^-1 (ODQ; 10^–5 mol l^–1) completely abolished the vasodilatory effect of the NO donor, sodium nitroprusside (SNP; 10^–4 mol l^–1). In addition, nicotine (3x10^–4 mol l^–1) mediated a vasodilation that was not affected by removal of the endothelium. The nicotine-mediated dilation was blocked by the NOS inhibitor, Nω-nitro-arginine (L-NNA; 10^–4 mol l^–1), and ODQ (10^–5 mol l^–1). More specifically, the neural NOS inhibitor, Nω-propyl-L-arginine (10^–5 mol l^–1), significantly decreased the dilation induced by nicotine (3x10^–4 mol l^–1). Furthermore, indomethacin (10^–5 mol l^–1) did not affect the nicotine-mediated dilation, suggesting that prostaglandins are not involved in the response. Finally, the calcium ionophore A23187 (3x10^–6 mol l^–1) caused an endothelium-dependent dilation that was abolished in the presence of indomethacin. We propose the absence of an endothelial NO system in eel vasculature and suggest that neurally derived NO contributes to the maintenance of vascular tone in this species. In addition, we suggest that prostaglandins may act as endothelially derived relaxing factors in A. australis.

Nitric oxide control of large veins in the toad Bufo marinus

This study examined the nitric oxide (NO) control of the vascular smooth muscle of the ventral abdominal vein and vena cava of the toad, Bufo marinus, by using anatomical and physiological approaches. Nicotinamide adenine di-nucleotide phosphate-diaphorase histochemistry and immunohistochemistry using endothelial nitric oxide synthase (NOS) and neural NOS antibodies produced no evidence for endothelial NOS in the veins, but, neural NOS-immunoreactive perivascular nerves were present. Acetylcholine (10^–5 M) caused a vasodilation in both veins that was endothelium-independent, and which was blocked by the soluble guanylyl cyclase inhibitor, ODQ (10^–5 M). The NOS inhibitors, L-NNA (10^–4 M) and L-NAME (10^–4 M), did not significantly reduce the vasodilatory effect of acetylcholine in the veins; this suggested that the vasodilation was not due to NO. However, in the presence of phenoxybenzamine (10^–7–10^–8 M), L-NNA significantly reduced the vasodilatory effect of acetylcholine in the veins. This unusual response is due to phenoxybenzamine partially inactivating the muscarinic receptor pool in the veins. In addition, the neural NOS inhibitor, vinyl-L-NIO (10^–5 M), significantly reduced the acetylcholine-mediated vasodilation in the presence of phenoxybenzamine. The results show that in toad veins, nitrergic nerves rather than an endothelial NO system are involved in NO-mediated vasodilation.

Dual mechanisms for nitric oxide control of large arteries in the estuarine crocodile crocodylus porosus

n reptiles, accumulating evidence suggests that nitric oxide (NO) induces a potent relaxation in the systemic vasculature. However, very few studies have examined the source from which NO is derived. Therefore, the present study used both anatomical and physiological approaches to establish whether NO-mediated vasodilation is via an endothelial or neural NO pathway in the large arteries of the estuarine crocodile Crocodylus porosus. Specific endothelial nitric oxide synthase (NOS) staining was observed in aortic endothelial cells following nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and endothelial NOS immunohistochemistry (IHC), suggesting that an endothelial NO pathway is involved in vascular control. This finding was supported by in vitro organ bath physiology, which demonstrated that the relaxation induced by acetylcholine (10-5 mol l-1) was abolished in the presence of the NOS inhibitor, N-omega-nitro-L-arginine (L-NNA; 10-4 mol l-1), the soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10-5 mol l-1), or when the endothelium was removed. Interestingly, evidence for a neural NO pathway was also identified in large arteries of the crocodile. Neural NOS was located in perivascular nerves of the major blood vessels following NADPH-d histochemistry and neural NOS IHC and in isolated aortic rings, L-NNA and ODQ, but not the removal of the endothelium, abolished the relaxation effect of the neural NOS agonist, nicotine (3x10-4 mol l-1). Thus, we conclude that the large arteries of C. porosus are potentially regulated by NO-derived from both endothelial and neural NOS.

Egg-laying-hormone immunoreactivity in the neural ganglia and ovary of Haliotis asinina Linnaeus

Immunoreactivity against the abalone egg-laying hormone (aELH) was detected in the fine granules of type 1 and 2 neurosecretory (NS) cells, neurites in the neuropil, and blood sinuses in the connective tissue sheath of the cerebral, pleuropedal, and visceral ganglia of the tropical abalone, Haliotis asinina Linnaeus. The number of positive NS cells, and the intensity of staining in the ganglia, varied and might be related to the stage of ovarian cycle. At any stage, positive cells were most numerous in the pleuropedal, and least numerous in the visceral ganglion. In addition, several cells of the statocyst and associated nerves also exhibited the immunoreactivity. In the ovary, the most intense reactivity was detected in the follicular and granular cells adjacent to mature oocytes, in the trabeculae and the ovarian capsule. The cytoplasm of mature oocytes was also moderately stained. The results indicate that the cerebral, pleuropedal, and visceral ganglia are the main sites of aELH-producing cells. The ovary may also produce aELH locally.

Carpal tunnel syndrome: diagnostic usefulness of sonography

PURPOSE: To prospectively evaluate accuracy of sonography for diagnosis of carpal tunnel syndrome (CTS) in patients clinically suspected of having the disease in one or both hands.
MATERIALS AND METHODS: A prospective cohort of 133 patients suspected of having CTS were referred to a teaching hospital between October 2001 and June 2002 for electrodiagnostic study. One hundred twenty patients (98 women, 22 men; mean age, 49 years; range, 19–83 years) underwent sonography within 1 week after electrodiagnostic study. Radiologist was blinded to electrodiagnostic study results. Seventy-five patients had bilateral symptoms; 23 patients, right-hand symptoms; and 22 patients, left-hand symptoms (total, 195 symptomatic hands). Cross-sectional area of median nerve was measured at three levels: immediately proximal to carpal tunnel inlet, at carpal tunnel inlet, and at carpal tunnel outlet. Flexor retinaculum was used as a landmark to margins of carpal tunnel. Optimal threshold levels (determined with classification and regression tree analysis) for areas proximal to and at tunnel inlet and at tunnel outlet were used to discriminate between patients with and patients without disease. Sensitivity, specificity, and false-positive and false-negative rates were derived on the basis of final diagnosis, which was determined with clinical history and electrodiagnostic study results as reference standard.
RESULTS: For right hands, sonography had sensitivity of 94% (66 of 70); specificity, 65% (17 of 26); false-positive rate, 12% (nine of 75); and false-negative rate, 19% (four of 21) (cutoff, 0.09 cm2 proximal to tunnel inlet and 0.12 cm2 at tunnel outlet). For left hands, sensitivity was 83% (53 of 64); specificity, 73% (24 of 33); false-positive rate, 15% (nine of 62); and false-negative rate, 31% (11 of 35) (cutoff, 0.10 cm2 proximal to tunnel inlet).
CONCLUSION: Sonography is comparable to electrodiagnostic study in diagnosis of CTS and should be considered as initial test of choice for patients suspected of having CTS.

The presence of GABA in gastropod mucus and its role in inducing larval settlement

Chemical substances that induce larval settlement have been the focus of many gastropod studies due to the importance of wild stock recruitment and production within aquaculture facilities. Gamma-aminobutyric acid (GABA), GABA analogs, and GABA-mimetics associated with certain crustose coralline algae (CCA), are known to induce larval settlement in commercial abalone (Haliotis) species, and other gastropods. Furthermore, mucus secreted from these gastropods has been shown to induce larval settlement, but the stimulatory components of mucus have not been thoroughly investigated. We now present data confirming that GABA is the settlement-inducing effector molecule contained within abalone mucus. To do this, we initially generated anti-GABA for use in immunoenzyme and immunofluorescent microscopy. Using these techniques GABA was identified in the nerves and epithelial cells of the foot, including mucus. Dried mucus samples subject to HPLC analysis revealed a mean concentration of 0.68 mM GABA after sample rehydration. The presence of GABA in these samples was confirmed by time-of-flight mass spectroscopy (TOF-MS). In addition, GABA was detected in the mucus of several abalone species and other gastropods by immunocytochemistry. Subsequent bioassays using both dry and fresh mucus strongly promoted induction of larval settlement.

Neurally-derived nitric oxide regulates vascular tone in pulmonary and cutaneous arteries of the toad, Bufo marinus

In this study, the role of nitric oxide (NO) in regulation of the pulmocutaneous vasculature of the toad, Bufo marinus was investigated. In vitro myography demonstrated the presence of a neural NO signaling mechanism in both arteries. Vasodilation induced by nicotine was inhibited by the soluble guanylyl cyclase (GC) inhibitor, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, and the NO synthase (NOS) inhibitor, N^ω-nitro-_L-arginine (_L-NNA). Removal of the endothelium had no significant effect on the vasodilation. Furthermore, pretreatment with N⁵-(1-imino-3-butenyl)-_L-ornithine (vinyl-_L-NIO), a more specific inhibitor of neural NOS, caused a significant decrease in the nicotine-induced dilation. In the pulmonary artery only, a combination of _L-NNA and the calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP(_8-37), completely blocked the nicotine-induced dilation. In both arteries, the vasodilation was also significantly decreased by glibenclamide, an ATP-sensitive K⁺ (K⁺_ATP) channel inhibitor. Levcromakalim, a K⁺_ATP channel opener, caused a dilation that was blocked by glibenclamide in both arteries. In the pulmonary artery, NO donor-mediated dilation was significantly decreased by pretreatment with glibenclamide. The physiological data were supported by NADPH-diaphorase histochemistry and immunohistochemistry, which demonstrated NOS in perivascular nerve fibers but not the endothelium of the arteries. These results indicate that the pulmonary and cutaneous arteries of B. marinus are regulated by NO from nitrergic nerves rather than NO released from the endothelium. The nitrergic vasodilation in the arteries appears to be caused, in part, via activation of K⁺_ATP channels. Thus, NO could play an important role in determining pulmocutaneous blood flow and the magnitude of cardiac shunting.

Vascular distribution of nitric oxide synthase and vasodilation in the Australian lungfish, Neoceratodus forsteri

The presence of nitric oxide synthase (NOS) and role of nitric oxide (NO) in vascular regulation was investigated in the Australian lungfish, Neoceratodus forsteri. No evidence was found for NOS in the endothelium of large and small blood vessels following processing for NADPH-diaphorase histochemistry. However, both NADPH-diaphorase histochemistry and neural NOS immunohistochemistry demonstrated a sparse network of nitrergic nerves in the dorsal aorta, hepatic artery, and branchial arteries, but there were no nitrergic nerves in small blood vessels in tissues. In contrast, nitrergic nerves were found in non-vascular tissues of the lung, gut and kidney. Dual-wire myography was used to determine if NO signalling occurred in the branchial artery of N. forsteri. Both SNP and SIN-1 had no effect on the pre-constricted branchial artery, but the particulate guanylyl cyclase (GC) activator, C-type natriuretic peptide, always caused vasodilation. Nicotine mediated a dilation that was not inhibited by the soluble GC inhibitor, ODQ, or the NOS inhibitor, L-NNA, but was blocked by the cyclooxygenase inhibitor, indomethacin. These data suggest that NO control of the branchial artery is lacking, but that prostaglandins could be endothelial relaxing factors in the vasculature of lungfish.

Human sympathetic nerve biology : parallel influences of stress and epigenetics in essential hypertension and panic disorder

Patients with panic disorder provide a clinical model of stress. On a "good day," free from a panic attack, they show persistent stress-related changes in sympathetic nerve biology, including abnormal sympathetic nerve single-fiber firing ("salvos" of multiple firing within a cardiac cycle) and release of epinephrine as a cotransmitter. The coreleased epinephrine perhaps originates from in situ synthesis by phenylethanolamine N-methyltransferase (PNMT). In searching for biological evidence that essential hypertension is caused by mental stress—a disputed proposition—we note parallels with panic disorder, which provides an explicit clinical model of stress: (1) There is clinical comorbidity; panic disorder prevalence is increased threefold in essential hypertension. (2) For both, epinephrine cotransmission is present in sympathetic nerves. (3) In panic disorder and essential hypertension, but not in health, single-fiber sympathetic nerve firing salvos occur. (4) Tissue nerve growth factor is increased in both conditions (nerve growth factor is a stress reactant). (5) There is induction of PNMT in sympathetic nerves. Essential hypertension exhibits a further manifestation of mental stress: there is activation of noradrenergic brain stem neurons projecting to the hypothalamus and amygdala. These pathophysiological findings strongly support the view that chronic mental stress is important in the pathogenesis of essential hypertension. A hypothesis now under test is whether in both disorders, under prevailing conditions of ongoing stress, PNMT induced in sympathetic nerves acts as a DNA methylase, causing the norepinephrine transporter (NET) gene silencing that is present in both conditions. PNMT can have an intranuclear distribution, binding to DNA. We have demonstrated that the reduced neuronal noradrenaline reuptake present in both disorders does have an epigenetic mechanism, with demonstrable reduction in the abundance of the transporter protein, the NET gene silencing being associated with DNA binding by the methylation-related inhibitory transcription factor MeCP2.