Simvastatin inhibits central sympathetic outflow in heart failure by a nitric-oxide synthase mechanism.

Our previous study demonstrated that oral treatment with simvastatin (SIM) suppressed renal sympathetic nerve activity (RSNA) in the rabbits with chronic heart failure (CHF). The purpose of this experiment was to determine the effects of direct application of SIM to the central nervous system on RSNA and its relevant mechanisms. Experiments were carried out on 21 male New Zealand White rabbits with pacing-induced CHF. The CHF rabbits received infusion of vehicle, SIM, or SIM + N(omega)-nitro-L-arginine methyl ester into the lateral cerebral ventricle via osmotic minipump for 7 days. We found that 1) in CHF rabbits, intracerebroventricular infusion of SIM significantly suppressed basal RSNA (1st day 69.5 +/- 8.9% maximum; 7th day 26.0 +/- 6.0% maximum; P < 0.05, n = 7) and enhanced arterial baroreflex function starting from the 2nd day and lasting through the following 5 days; 2) statin treatment significantly up-regulated neuronal nitric-oxide synthase (nNOS) protein expression in the rostral ventrolateral medulla (RVLM) (control, n = 6, 0.12 +/- 0.04; SIM-treated, n = 7, 0.31 +/- 0.05. P < 0.05); 3) in CATH.a neurons, incubation with SIM significantly up-regulated the nNOS mRNA expression, which was blocked by coincubation with mevalonate, farnesyl-pyrophosphate, or geranylgeranyl-pyrophosphate; and 4) incubation with Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide] significantly up-regulated nNOS mRNA expression in these neurons. These results suggest that central treatment with SIM decreased sympathetic outflow in CHF rabbits via up-regulation of nNOS expression in RVLM, which may be due to the inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase and a decrease in Rho kinase by SIM.

Distribution of nitric oxide synthase-containing ganglionic neuronal somata and postganglionic fibers in the rat kidney.

Nitric oxide synthase (NOS)-immunoreactive neurons were identified in the rat kidney by using an antibody against type Ia NOS and the avidin-biotin complex immunoperoxidase method in whole kidneys examined in 100 microns serial sections. The histochemical method for demonstration of the nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) was also used to characterize NOS-containing neurons. All somata showing NOS immunoreactivity also displayed NADPH-d activity. The greatest number of neuronal somata were observed in groups at the wall of the renal pelvis and in the angular space formed by the pole of the renal parenchyma and renal pelvic wall. They were also seen at the renal hilus close to the renal artery and along the interlobar vasculature. The size of the neuronal somata in the 35-day-old rat ranged from 13.6 to 34.8 microns, with a mean size of 21.52 +/- 4.81 microns. Seventy percent, however, ranged in size from 17.8 to 26.8 microns. The shape of the neuronal somata also varied, with the majority having an ovoid or round shape. The distribution of the postganglionic fibers was investigated by means of the camera lucida. Postganglionic fibers projected into the wall of the renal pelvis and/or to the interlobar arteries extending to the arcuate arteries and to the beginning of the afferent arterioles. The NOS-immunoreactive neurons may have a vasodilator and relaxing function on the renal pelvic wall and vasculature. In addition, the presence of NOS-containing nerve fibers in nerve bundles, which are known to have predominantly vasomotor and sensory fibers, suggest that they may have a possible modulatory role on renal neural function.

Sympathetic sprouting in the dorsal root ganglia of the injured peripheral nerve in a rat neuropathic pain model.

The extent of the sprouting of sympathetic postganglionic fibers in the dorsal root ganglion (DRG) and the peripheral nerves was examined in neuropathic rats at different postoperative times. After the L5 and L6 spinal nerves were ligated on one side, three different pain behavior tests (representing mechanical allodynia, cold allodynia, ongoing pain exacerbated by cold stress) were performed at various time intervals. The sympathetic postganglionic fibers were visualized by immunostaining with antibodies to tyrosine hydroxylase (TH). In the neuropathic rats, all three pain behaviors were fully developed within 3 days after the surgery, maintained up to 2 weeks, and then started to decline gradually afterward. At 20 weeks after neuropathic surgery, pain behaviors were reduced significantly compared to the peak response, but were still higher than the presurgery levels. Sympathectomy, performed 4 days after neuropathic surgery, almost completely abolished the signs of mechanical allodynia and ongoing pain behaviors, and it reduced the behaviors of cold allodynia to approximately half. The numerical density of sympathetic fibers in the DRG of an injured segment was significantly higher at 1, 4, and 20 weeks after neuropathic surgery as compared to the normal, suggesting that there is sprouting of sympathetic fibers in the DRG after peripheral nerve injury. Sprouting of sympathetic fibers in the DRG was extensive as early as 2 days after the spinal nerve ligation, and the sprouted fibers were almost completely eliminated after sympathectomy. The data suggest that sympathetic innervation of the DRG may play an important role in the development and maintenance of sympathetically maintained neuropathic pain.

Sympathetic innervation of the upper and lower regions of the uterus and cervix in the rat have different origins and routes.

The origins and routes of the postganglionic sympathetic nerve supply to the upper and lower uterus and to the cervix were investigated in the rat by using denervation procedures combined with immunohistochemistry and retrograde tracing. The sympathetic nerve fibers of the upper part of the uterus arise from the ovarian plexus nerve. They mainly originate (90%) from neurons of the suprarenal ganglia (SRG) and of the T10 to L3 ganglia of the paravertebral sympathetic chain. Fluoro-Gold injections into different regions of the upper uterus showed that the SRG neurons mainly provide innervation to the tubal extremity (52%) rather than to the uterine portion below this area (26%). Very few neurons of the celiac ganglion or the aorticorenal ganglia participated in this innervation. Most of the sympathetic innervation of the lower uterus and the cervix (90%) originates from neurons of the paravertebral ganglia T13 to S2, principally at the L2-L4 levels. By using immunocytochemistry, we show that very few tyrosine hydroxylase-positive neurons of the pelvic plexus project to these areas, where they represent only 3% of the sympathetic nerve supply. Again, very few neurons of the inferior mesenteric ganglion (IMG) supply the lower uterus and the cervix. The comparison between retrograde tracing experiments in intact animals and after the removal of the IMG shows that very few sympathetic postganglionic axons from the paravertebral chain pass through the IMG to reach the lower uterus and the cervix. In contrast, these axons mainly project to splanchnic nerves bypassing the IMG to connect with the hypogastric nerves. In addition, some axons supplying the lower uterus follow the superior vesical arteries and then reach the organ. Taken together, these results show that the upper region of the uterus receives a sympathetic innervation that is different in origin and route from that of the lower uterus and the cervix. Such a marked region-specific innervation suggests that nerve control of the myometrial activity may be functionally different between the oviduct and the cervical ends of the uterus.

TRPC6 immunoreactivity is colocalized with neuronal nitric oxide synthase in extrinsic fibers innervating guinea pig intrinsic cardiac ganglia.

Tachykinins depolarize guinea pig intracardiac neurons by activating nonselective cationic channels. Recently, members of the transient receptor potential family of membrane channels (TRPC) have been implicated in the generation of G protein-coupled receptor-activated nonselective cationic currents. We have investigated whether guinea pig cardiac neurons exhibit immunoreactivity to TRPC. Our results showed that nerve fibers within guinea pig intrinsic cardiac ganglia exhibited immunoreactivity to TRPC6. After culture of cardiac ganglia whole-mount explants for 72 hours, the TRPC6-IR fiber networks were absent. Therefore, the TRPC6-IR fibers were derived from sources extrinsic to the heart. A small percentage ( approximately 3%) of intracardiac neurons also exhibited TRPC6 immunoreactivity in control preparations, and the percentage of cells exhibiting TRPC6 immunoreactivity was not changed following explant culture for 72 hours. The few intrinsic TRPC6-IR neurons also exhibited nitric oxide synthase (NOS) immunoreactivity, indicating that they were nitrergic as well. We compared the immunohistochemical staining patterns of TRPC6-IR fibers with the staining patterns of a number of other neurotransmitters or neurotransmitter synthetic enzymes that mark specific extrinsic inputs to the intrinsic cardiac ganglia. The TRPC6-IR fibers were not immunoreactive for choline acetyltransferase, tyrosine hydroxylase, or substance P. However, the TRPC6-IR fibers exhibited immunoreactivity to neuronal NOS. Therefore, we propose that the TRPC6-IR fibers within the guinea pig intrinsic cardiac ganglia are vagal sensory fibers that also contain NOS. We found, in support of this conclusion, that TRPC6-IR cells were also present in sections of nodose ganglia.

In vivo observation of a non-noradrenergic regulation of arylalkylamine N-acetyltransferase gene expression in the rat pineal complex.

The rodent pineal gland is the end point of several peripheral and central fibers innervating the superficial and deep parts of the gland. Up to now, only the sympathetic transmitter norepinephrine is thought to regulate melatonin synthesis, although numerous biochemical experiments have reported in vitro effects of various transmitters on melatonin synthesis. To find out whether there is non-noradrenergic regulation of in vivo pineal metabolism, the mRNA encoding the enzyme arylalkylamine N-acetyltransferase was studied using the highly sensitive technique of in situ hybridization. The existence of a marked nocturnal increase of arylalkylamine N-acetyltransferase mRNA in the superficial pineal gland was confirmed. Interestingly and for the first time, a similar daily variation was observed in the deep pineal. After removal of superior cervical ganglia, the daily rhythm in arylalkylamine N-acetyltransferase mRNA was abolished in both the superficial and deep pineal indicating that the rhythm is driven by sympathetic input in the entire pineal complex. Interestingly, the remaining arylalkylamine N-acetyltransferase mRNA level in the pineal of day- and night-time ganglionectomized rats was significantly higher than in the pineal of day-time intact animals. These data reveal a sympathetic-dependent day-time inhibition of arylalkylamine N-acetyltransferase gene expression. In addition, the day-time pineal arylalkylamine N-acetyltransferase mRNA expression in ganglionectomized rats persisted after adrenal gland removal but was reduced by 50% after propranolol injection. These results indicate that arylalkylamine N-acetyltransferase mRNA in ganglionectomized rats is not induced by circulating catecholamines and may be caused by both a centrally originated norepinephrine, as already suggested, and other non-adrenergic transmitter(s). In conclusion, this work shows that norepinephrine drives the nocturnal increase of arylalkylamine N-acetyltransferase gene expression both in the superficial and deep pineal and strongly suggests that other neurotransmitters are involved in day-time inhibition and night-time stimulation of pineal metabolism.

Morphological and functional evidence against a sensory and sympathetic origin of nitric oxide synthase-containing nerves in the rat lower urinary tract.

To establish which type of nerves (parasympathetic, sympathetic or sensory) produce nitric oxide in the rat lower urinary tract, chemical denervation of primary afferents and sympathetic nerves was carried out by systemic treatment with capsaicin and 6-hydroxydopamine, respectively, followed by identification of neuronal nitric oxide synthase immunoreactivity. Functional in vitro studies were also performed to examine whether the synthesis and release of nitric oxide was affected following treatment with the respective neurotoxins. Nerve fibres immunoreactive for substance P and calcitonin gene-related peptide were found in control tissue, but could not be detected following capsaicin treatment. In comparison, nitric oxide synthase-immunoreactive fibres appeared to be unaffected by capsaicin treatment. Administration of 6-hydroxydopamine resulted in a complete disappearance of tyrosine hydroxylase-immunoreactive nerves, whereas nitric oxide synthase-containing nerve fibres did not appear to be affected by the treatment. In ultrastructural studies, nitric oxide synthase immunoreactivity, as studied by colloidal gold particles, was found in the axoplasm and not in association with intraneuronal structures or synaptic vesicles. Gold particles representing substance P immunoreactivity were seen as clusters associated with large granular vesicles. In consecutive sections of nerve fibres, substance P and nitric oxide synthase were not found in the same axon profile. In functional studies on urethral tissue, application of capsaicin (1 microM) produced a long-lasting relaxation. The nitric oxide synthase inhibitor NG-nitro-L-arginine (0.1 mM) had no effect on this response. Systemic treatment with capsaicin or 6-hydroxydopamine had no effect on nerve-evoked, nitric oxide-mediated relaxations. The data suggest that nitric oxide synthase-containing nerves in the rat lower urinary tract do not belong to nerve populations sensitive to either the sympathetic neurotoxin, 6-hydroxydopamine, or the sensory neurotoxin, capsaicin.

Presence of neuronal nitric oxide synthase in autonomic and sensory ganglion neurons innervating the lacrimal glands of the cat: an immunofluorescent and retrograde tracer double-labeling study.

It is generally considered that parasympathetic postganglionic nerve fibers innervating the lacrimal gland (LG) arise from the pterygopalatine ganglion (PPG), while sympathetic and sensory innervations arise from the superior cervical ganglion (SCG) and trigeminal ganglion (TG), respectively. Recently, we reported for the first time that the parasympathetic innervation of the cat LG was also provided by the otic ganglion (OG) and ciliary ganglion (CG), and that the sensory innervation was also provided by the superior vagal ganglion (SVG) and superior glossopharyngeal ganglion (SGG). To determine if nitric oxide (NO) is a neurotransmitter of the autonomic and sensory neurons innervating the LG, we injected the cholera toxin B subunit (CTB) as a retrograde tracer into the cat LG, and used double-labeling fluorescent immunohistochemistry for CTB and nitric oxide synthase (NOS). We found that NOS-/CTB-immunofluorescent double-labeled perikarya were localized in the PPG, OG, TG, SVG and SGG, but not in the CG and SCG. The highest numbers of NOS-/CTB-immunofluorescent double-labeled neurons were found in the PPG and TG. In addition, we examined the presence of nitrergic nerve fibers in the LG using NADPH-d histochemistry and found that a large amount of NADPH-d-stained nerve fibers were distributed around the glandular acini and in the walls of glandular ducts and blood vessels. This study provides the first direct evidence showing that NO may act as a neurotransmitter or modulator involved in the parasympathetic and sensory regulation of lacrimal secretion and blood circulation, but may not be implicated in the sympathetic control of LG activities, and that nitrergic nerve fibers in the LG arise mainly from parasympathetic postganglionic neurons in the PPG and sensory neurons in the TG. The present results suggest that NO plays an important role in the regulation of LG activities.

Guinea-pig sympathetic postganglionic neurones contain haem oxygenase-2.

HAEM oxygenase-2 (HO-2) is the neuronal isoform of the only known mammalian enzyme generating the new transmitter candidate, carbon monoxide. Its distribution was investigated in the sympathetic nervous system. A 36 kDa HO-2 immunoreactive protein was identified in the particulate fraction of stellate ganglion and cerebellum in Western blots. Immunohistochemically, all noradrenergic and non-noradrenergic postganglionic neurones were HO-2 immunoreactive in prevertebral ganglia and cervical, thoracic, and lumbar sympathetic chain ganglia. Neither postganglionic nerve branches nor noradrenergic perivascular terminals were HO-2 immunoreactive under control conditions. However, accumulation of HO-2 immunoreactivity was found in noradrenergic axons in explanted sciatic nerves in which axonal transport was interrupted by crushing. We conclude that HO-2 is ubiquitous in perikarya of postganglionic sympathetic neurones, is subjected to axonal transport but does not accumulate at sympathetic neuroeffector junctions. This distribution favours a general role of HO-2 in sympathetic neuronal metabolism rather than a specific association with transmission at autonomic neuroeffector junctions.

NADPH-diaphorase activity and its colocalization with transmitters and neuropeptides in the postganglionic neurons of the rat superior cervical ganglion.

NADPH-diaphorase activity (NADPH-DA), a marker of neural nitric oxide synthase, was found in many postganglionic nerve cell bodies in the adult rat superior cervical ganglion (SCG) after colchicine treatment, postganglionic nerve trunk ligation or ganglion culture. NADPH-DA colocalized with immunoreactivity to tyrosine hydroxylase (TH), serotonin, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), methionine-enkephalin and somatostatin. Almost all cells showing NADPH-DA were TH-immunoreactive, although several TH-immunoreactive cells lacked NADPH-DA. While suggesting that nitric oxide has an important role in the neuronal modulation in the synaptic transmission in the rat SCG, our results point out that nitric oxide synthesis is confined to a subpopulation of ganglion neurons. Our findings confirm the idea that the superior cervical ganglion consists of several subpopulations in which noradrenaline is colocalized with other transmitter or neuropeptide. Only about one-fourth of serotonin-immunoreactive neurons contained NADPH-DA. Similarly, the neuropeptides studied showed only partial colocalization with NADPH-DA. Our results thus suggest that nitric oxide is not associated with any particular transmitter or peptide.

Nitric oxide synthase immunoreactive neurons in the rat kidney.

The presence of neurons with nitric oxide synthase (NOS) immunoreactivity was investigated in the rat kidney. Whole kidneys were examined by means of serial sections. The indirect immunocytochemical technique using polyclonal antibody raised against rat brain type Ia NOS and the histochemical technique for nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase are used in this study. NOS-immunoreactive (NOS-IR) neurons varied in size and were observed: (1) associated with nerve bundles at the hilus of the kidney, (2) in the proximity of the lower or middle portion of the interlobar arteries, and (3) on the wall of the renal pelvis. We are presenting anatomic evidence for the presence of neurons in the rat kidney. Their location is consistent with the existence of a parasympathetic innervation of the rat kidney.

Immunohistochemical determination of the sympathetic pathway in the orbit via the cranial nerves in humans.

OBJECT: The present study was undertaken to elucidate the extent and precise distribution of the postganglionic sympathetic fibers in the cranial nerves projecting to the orbit and to reconstruct sympathetic routes in the orbit in humans. For this purpose, the authors made an immunohistochemical determination of the sympathetic fibers by using an antibody against norepinephrine-synthetic enzyme, tyrosine hydroxylase (TH). METHODS: Specimens containing the orbit and the cavernous sinus were obtained from formalin-fixed human cadavers. First, it was confirmed that the superior cervical ganglion contained strongly immunostained TH-positive neuronal cell bodies and fibers. After careful dissection of the cranial nerves projecting to the orbit, different segments of each cranial nerve were processed for immunohistochemical analysis for TH. All of the intraorbital cranial nerves contained TH-positive sympathetic fibers, although the amounts were very different in each cranial nerve. At the proximal site of the common tendinous ring, TH-positive fibers were found mainly in the abducent and trochlear nerves. At the distal site of this ring, TH-positive fibers were lost or markedly reduced in number in the abducent and trochlear nerves and were distributed mostly in the ophthalmic and oculomotor nerves. Among the cranial nerves projecting to the orbit, the ophthalmic nerve and its bifurcated nerves--frontal, lacrimal, and nasociliary--contained numerous TH-positive fibers. CONCLUSIONS: The authors conclude that the postganglionic sympathetic fibers are distributed to all cranial nerves projecting to the orbit and that the ophthalmic nerve provides a major sympathetic route in the orbital cavity in humans.

Choline acetyltransferase immunoreactive sympathetic ganglion cells in a teleost, Stephanolepis cirrhifer.

The present study showed neurons immunoreactive for choline acetyltransferase (ChAT) in the cranial sympathetic ganglia lying close to the trigeminal-facial nerve complex of the filefish. In these ganglia, less than 1% of ganglion cells were positive for choline acetyltransferase. Choline acetyltransferase-positive neurons were significantly larger than the randomly sampled neurons in this ganglion. The majority of choline acetyltransferase-positive neurons were negative for tyrosine hydroxylase, but many of them were positive for galanin (GAL). Some neurons were positive for both choline acetyltransferase and tyrosine hydroxylase, but these neurons were rarely immunoreactive for dopamine beta hydroxylase, suggesting that they are not adrenergic. In the cranial sympathetic ganglia and the celiac ganglia, many nerve fibers immunoreactive for galanin were seen, and varicose terminals were in contact selectively with neurons negative for both choline acetyltransferase and tyrosine hydroxylase, but not with those positive for choline acetyltransferase or tyrosine hydroxylase. Nerve fibers immunoreactive for choline acetyltransferase were found to be present in contact with the deep layer of chromatophores, which was observed only in the labial region. These results suggest that cholinergic postganglionic neurons are present in the filefish cranial sympathetic ganglia, and that they also contain galanin. As few cholinergic sympathetic neurons express tyrosine hydroxylase and none express dopamine beta hydroxylase, they are unlikely to synthesize noradrenaline or adrenaline.

Site-dependent differences in density of sympathetic nerve fibers in muscle-innervating nerves of the human head and neck.

The autonomic nerve supply of skeletal muscle has become a focus of interest because it is closely related to the adaptation of energy metabolism with aging. We have performed an immunohistochemistry study on tyrosine hydroxylase (TH) and neuronal nitric oxide synthase (nNOS) using specimens obtained from ten selected elderly cadavers (mean age 83.3 years) in which we examined muscle-innervating nerves (abbreviated ''muscle-nerves'' hereafter) of ten striated muscles (soleus, infraspinatus, extra-ocular inferior rectus, lateral rectus, superior obliquus, temporalis, orbicularis oculi, posterior cricoarytenoideus, trapezius and genioglossus) and, as a positive control, the submandibular ganglion. We found that the extra-ocular muscles received no or very few TH-positive nerve fibers. Muscle-nerves to the other head and neck muscles contained a few or several TH-positive fibers per section, but their density (proportional area of TH-positive fibers per nerve cross-section) was one-half to one-third of that in nerves to the soleus or infraspinatus. We did not find nNOS-positive fibers in any of these muscle-nerves. In the head and neck muscles, with the exception of those of the tongue, there appeared to be very few TH-positive nerve fibers along the feeding artery. Consequently, the head and neck muscles seemed to receive much fewer sympathetic nerves than limb muscles. There was no evidence that nNOS-positive nerves contributed to vasodilation of feeding arteries in striated muscles. This site-dependent difference in sympathetic innervation would reflect its commitment to muscle activity. However, we did not find any rules determining the density of nerves according to muscle fiber type and the mode of muscle activity.

Effect of pioglitazone on muscle sympathetic nerve activity in type 2 diabetes mellitus with α-glucosidase inhibitor.

Activation of the sympathetic nervous system is augmented in patients with type 2 diabetes mellitus (DM). Pioglitazone, an anti-diabetic drug, improves insulin resistance, but its influence on sympathetic nerve activity is not clear. To identify the relationship between insulin resistance and sympathetic activity, we examined muscle sympathetic nerve activity (MSNA) in controlled type 2 DM patients with alpha-glucosidase inhibitor (GI). We measured MSNA and calculated homeostasis model assessment of insulin resistance index (HOMA-IR) in twelve DM patients treated with alpha-GI and thirteen age-matched healthy subjects. In DM patients with alpha-GI, all parameters were reexamined after three months of treatment with pioglitazone. MSNA and HOMA-IR were significantly greater in DM patients with alpha-GI compared to healthy subjects. Hemoglobin A1c did not differ in DM patients before and after pioglitazone. However, pioglitazone significantly decreased MSNA in DM patients compared with alpha-GI (21.7±5.2 vs. 32.0±6.8 burst/min, p<0.01). Furthermore, MSNA level in pioglitazone was similar to that in healthy subjects. HOMA-IR significantly decreased after pioglitazone, and a significant relationship was found between the absolute change in MSNA and HOMA-IR (r=0.65, p<0.05). These results suggest that improved insulin resistance with pioglitazone provides an additional effect on inhibition of sympathetic nerve activity.

Localization of NADPH oxidase in sympathetic and sensory ganglion neurons and perivascular nerve fibers.

Superoxide anion (O(2)(-*)) production was previously reported to be increased in celiac ganglia (CG) during DOCA-salt hypertension, possibly via activation of the reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. This suggested a role for neuronal NADPH oxidase in autonomic neurovascular control. However, the expression and localization of NADPH oxidase in the peripheral neurons are not fully known. The purpose of this study was to examine the subcellular localization of NADPH oxidase in sympathetic and sensory ganglion neurons and perivascular nerve fibers. In rat CG, p22(phox) and neuropeptide Y (NPY) were colocalized in all neurons. P22(phox) was also localized to dorsal root ganglia (DRG) neurons that contain calcitonin gene related peptide (CGRP). In mesenteric arteries, p22(phox) and p47(phox) were colocalized with NPY or CGRP in perivascular nerve terminals. A similar pattern of nerve terminal staining of p22(phox) and p47(phox) was also found in cultured CG neurons and nerve growth factor (NGF)-differentiated PC12 cells. These data demonstrate a previously uncharacterized localization of NADPH oxidase in perivascular nerve fibers. The presence of a O(2)(-*)-generating enzyme in close vicinity to the sites of neurotransmitter handling in the nerve fibers suggests the possibility of novel redox-mediated mechanisms in peripheral neurovascular control.

TH and NPY in sympathetic neurovascular cultures: role of LIF and NT-3.

The sympathetic nervous system is an important determinant of vascular function. The effects of the sympathetic nervous system are mediated via release of neurotransmitters and neuropeptides from postganglionic sympathetic neurons. The present study tests the hypothesis that vascular smooth muscle cells (VSM) maintain adrenergic neurotransmitter/neuropeptide expression in the postganglionic sympathetic neurons that innervate them. The effects of rat aortic and tail artery VSM (AVSM and TAVSM, respectively) on neuropeptide Y (NPY) and tyrosine hydroxylase (TH) were assessed in cultures of dissociated sympathetic neurons. AVSM decreased TH (39 +/- 12% of control) but did not affect NPY. TAVSM decreased TH (76 +/- 10% of control) but increased NPY (153 +/- 20% of control). VSM expressed leukemia inhibitory factor (LIF) and neurotrophin-3 (NT-3), which are known to modulate NPY and TH expression. Sympathetic neurons innervating blood vessels expressed LIF and NT-3 receptors. Inhibition of LIF inhibited the effect of AVSM on TH. Inhibition of neurotrophin-3 (NT-3) decreased TH and NPY in neurons grown in the presence of TAVSM. These data suggest that vascular-derived LIF decreases TH and vascular-derived NT-3 increases or maintains NPY and TH expression in postganglionic sympathetic neurons. NPY and TH in vascular sympathetic nerves are likely to modulate NPY and/or norepinephrine release from these nerves and are thus likely to affect blood flow and blood pressure. The present studies suggest a novel mechanism whereby VSM would modulate sympathetic control of vascular function.

Sympathetic control of heart rate in nNOS knockout mice.

Inhibition of neuronal nitric oxide synthase (nNOS) in cardiac postganglionic sympathetic neurons leads to enhanced cardiac sympathetic responsiveness in normal animals, as well as in animal models of cardiovascular diseases. We used isolated atria from mice with selective genetic disruption of nNOS (nNOS(-/-)) and their wild-type littermates (WT) to investigate whether sympathetic heart rate (HR) responses were dependent on nNOS. Immunohistochemistry was initially used to determine the presence of nNOS in sympathetic [tyrosine hydroxylase (TH) immunoreactive] nerve terminals in the mouse sinoatrial node (SAN). After this, the effects of postganglionic sympathetic nerve stimulation (1-10 Hz) and bath-applied norepinephrine (NE; 10(-8)-10(-4) mol/l) on HR were examined in atria from nNOS(-/-) and WT mice. In the SAN region of WT mice, TH and nNOS immunoreactivity was virtually never colocalized in nerve fibers. nNOS(-/-) atria showed significantly reduced HR responses to sympathetic nerve activation and NE (P < 0.05). Similarly, the positive chronotropic response to the adenylate cyclase activator forskolin (10(-7)-10(-5) mol/l) was attenuated in nNOS(-/-) atria (P < 0.05). Constitutive NOS inhibition with L-nitroarginine (0.1 mmol/l) did not affect the sympathetic HR responses in nNOS(-/-) and WT atria. The paucity of nNOS in the sympathetic innervation of the mouse SAN, in addition to the attenuated HR responses to neuronal and applied NE, indicates that presynaptic sympathetic neuronal NO does not modulate neuronal NE release and SAN pacemaking in this species. It appears that genetic deletion of nNOS results in the inhibition of adrenergic-adenylate cyclase signaling within SAN myocytes.

Quantitative analysis of the sympathetic innervation of the rat knee joint.

Retrograde tracing with Fluoro-Gold (FG) was used to identify the complete population of knee joint sympathetic postganglionic efferents in the lumbar sympathetic chain of adult female Wistar rats. In 6 rats, the total number and distribution of FG-labelled neurons in the lumbar sympathetic chain was determined. The rat knee joint is supplied by an average of 187+/-57 sympathetic afferents with the majority at the L3 and L4 levels. Immunohistochemistry using antibodies specific for tyrosine hydroxylase (TH), somatostatin (SS) or vasoactive intestinal polypeptide (VIP) revealed that 33 % of knee joint sympathetic afferents contained TH, 42 % contained VIP, and none contained somatostatin. Retrograde tracing with FG provided accurate and reproducible labelling of the joint-innervating subpopulation of sympathetic efferent neurons. This model lends itself to the further study of the molecular responses of this neuronal population in the various disorders and conditions affecting joints.

Chronic transection of post-ganglionic parasympathetic and nasociliary nerves does not affect local cerebral blood flow in the rat.

The role of post-ganglionic parasympathetic nerve fibers from the sphenopalatine ganglion and nasociliary nerve fibers from the trigeminal ganglion in the regulation of basal cerebral blood flow (CBF) was examined using rats, which had been divided into three groups; a sham group, a denervation group and a denervation+NG-monomethyl-L-arginine (L-NMMA) group. In the denervation and denervation+L-NMMA groups, unilateral chronic transection of the above nerve fibers had been performed at the ethmoidal foramen (EF) for 2 weeks. In the sham group, the above nerve fibers were only exposed at EF and not severed 2 weeks before the CBF measurement. Local CBF was measured by the [14C]iodoantipyrine autoradiographic method after intravenous administration of saline in the sham and denervation groups or L-NMMA (30 mg/kg) in the denervation+L-NMMA group. No significant difference in CBF was noted on each side in any of the regions between the sham and denervation groups. L-NMMA induced a significant reduction in local CBF on either side in each brain region. Neither the animals which were administered saline nor those with L-NMMA showed any side-to-side differences in local CBF in any of the brain regions examined. These findings suggest that the perivascular nerve fibers running through the EF, which are known to contain substantial nitric oxide synthase (NOS), may not play a pivotal role in the regulation of basal CBF. The reduction in CBF induced by the acute administration of L-NMMA was not affected by the chronic denervation of the above NOS-containing perivascular nerves.