Pflugers Arch - Eur J Physiol DOI 10.1007/s00424-014-1621-0

INVITED REVIEW

Recent advances in research on nitrergic nerve-mediated vasodilatation Noboru Toda & Tomio Okamura

Received: 10 August 2014 / Revised: 25 September 2014 / Accepted: 29 September 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Cerebral vascular resistance and blood flow were widely considered to be regulated solely by tonic innervation of vasoconstrictor adrenergic nerves. However, pieces of evidence suggesting that parasympathetic nitrergic nerve activation elicits vasodilatation in dog and monkey cerebral arteries were found in 1990. Nitric oxide (NO) as a neurotransmitter liberated from parasympathetic postganglionic neurons decreases cerebral vascular tone and resistance and increases cerebral blood flow, which overcome vasoconstrictor responses to norepinephrine liberated from adrenergic nerves. Functional roles of nitrergic vasodilator nerves are found also in peripheral vasculature, including pulmonary, renal, mesenteric, hepatic, ocular, uterine, nasal, skeletal muscle, and cutaneous arteries and veins; however, adrenergic nerve-induced vasoconstriction is evidently greater than nitrergic vasodilatation in these vasculatures. In coronary arteries, neurogenic NO-mediated vasodilatation is not clearly noted; however, vasodilatation is induced by norepinephrine released from adrenergic nerves that activates β1-adrenoceptors. Impaired actions of NO liberated from the endothelium and nitrergic neurons are suggested to participate in cerebral hypoperfusion, leading to brain dysfunction, like that in Alzheimer’s disease. Nitrergic neural dysfunction participates in impaired circulation in peripheral organs and tissues and also in systemic blood pressure increase. NO and vasodilator peptides, as sensory neuromediators, are involved in neurogenic vasodilatation in the skin. Functioning of nitrergic vasodilator nerves is evidenced not only in a variety of mammals, N. Toda (*) Toyama Institute for Cardiovascular Pharmacology Research, 7-13, 1-Cho-me, Azuchi-machi, Chuo-ku, Osaka 541-0052, Japan e-mail: [email protected] N. Toda : T. Okamura Department of Pharmacology, Shiga University of Medical Science, Seta, Otsu 520-2192, Japan

including humans and monkeys, but also in non-mammals. The present review article includes recent advances in research on the functional importance of nitrergic nerves concerning the control of cerebral blood flow, as well as other regions, and vascular resistance. Although information is still insufficient, the nitrergic nerve histology and function in vasculatures of non-mammals are also summarized. Keywords Nitrergic nerve . Vasodilatation . Nitric oxide . Cerebral artery . Peripheral arteries . Nitrergic and adrenergic nerve interaction Abbreviations ADMA Asymmetric dimethylarginine CGRP Calcitonin gene-related peptide eNOS Endothelial NOS 5-HT 5-Hydroxytryptamine L-NA NG-nitro-L-arginine NADPH Reduced nicotinamide adenine dinucleotide phosphate L-NAME L-NA methylester 7-NI 7-Nitroindazole G L-NMMA N -monomethyl-L-arginine nNOS Neuronal NOS NO Nitric oxide NOS Nitric oxide synthase PIN Protein inhibitor of nNOS L-SMTC S-methyl-L-thiocitrulline SHR Spontaneously hypertensive rats

Introduction The endothelium-derived relaxing factor discovered by Furchgott and Zawadzki [29] was identified to be nitric oxide

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(NO) liberated from endothelial cells [28, 68]. NO plays quite important roles in circulation, including vasodilatation, increasing blood flow, decreasing vascular resistance, lowering blood pressure, inhibition of platelet adhesion, and migration, and reduction of smooth muscle proliferation. The discovery of NO synthase (NOS) inhibitors, such as N-monomethyl-L-arginine (LNMMA) [69], Nnitro- L -arginine ( L -NA), and L -NA methylester ( L NAME), has enabled researchers to determine the involvement of NO in vascular responses to physiological and chemical stimuli. In addition, neuronal NOS (nNOS)-selective inhibitors, such as 7-nitroindazole (7NI) [58], S-methyl-L-thiocitrulline (L-SMTC; IC50 value of 0.047 nM) [30], and propyl-L-arginine (IC50 value of 57 nM) [128], have made it possible to discriminate responses mediated via NO derived from endothelial cells. Electrical nerve stimulation or nicotine, a chemical stimulant of autonomic neurons, induces vasodilatation (Fig. 1) only in isolated canine cerebral and coronary arteries but vasoconstriction in other arteries [111]. The coronary arterial dilatation induced by nerve stimulation is abolished by treatment with β-adrenoceptor antagonists, whereas neurogenic cerebral arterial dilatation is not influenced by β-blockers and cyclooxygenase inhibitors or in preparations desensitized to vasodilator polypeptides, such as vasoactive intestinal polypeptide, calcitonin gene-related polypeptide (CGRP), and substance P. Functional roles of non-adrenergic, non-cholinergic, and non-peptidergic vasodilator nerves were first evidenced in isolated canine [92] and cat cerebral arteries [51], but the mechanisms of action were not determined. Ten years later, it was found that the neurogenic cerebroarterial dilatation was abolished by the NOS inhibitor L-NMMA, but not D-NMMA, and treatment with L-arginine restored the dilator response [105, 106]. This parasympathetic, postganglionic nerve was named the “nitroxidergic nerve” [103] but later unified by the International Union of Pharmacological Society to the “nitrergic nerve.” Vasoconstriction in response to electrical nerve stimulation in other peripheral arteries, such as pulmonary, mesenteric, renal, ocular, skeletal muscle, etc., is reversed to vasodilatation after treatment with α-adrenoceptor antagonists or adrenergic neuron blockers, suggesting that these vasculatures are reciprocally innervated by adrenergic vasoconstrictor and nitrergic vasodilator nerves. This review article covers recent advances in research on the role of nitrergic nerves in the control of vascular tone, vascular resistance, and blood flow in cerebral and peripheral vasculatures in mammals. Differences in the neurogenic vasodilatation in various arteries are also compared. Although not sufficient, some information concerning nitrergic nerve function and histology in non-mammals is also included.

Cerebral blood vessels Nitrergic neurogenic vasodilatation is clearly elucidated in cerebral arteries in response to electrical stimulation or nicotine, whereas in peripheral arteries and veins, vasodilator responses are visible only when the tissues are treated with α-adrenoceptor blockers or adrenergic neuron blockers in various mammals [107]. There are functional and histological pieces of evidence indicating that human [94], monkey [106], dog [105], pig [50], bovine [7], guinea pig [43], and rat cerebral arteries [40] are innervated by nitrergic vasodilator nerves. Electrical stimulation of the pterygopalatine ganglion induces vasodilatation of middle cerebral and anterior cerebral arteries and also neighboring arterioles in anesthetized Japanese monkeys [110] (Fig. 2) and dogs [99]. Studies on anesthetized Japanese monkeys have demonstrated that relaxations in response to electrical nerve stimulation and nicotine were attenuated by 2×10−5 M 7-NI, a nNOS inhibitor, but the endothelium-dependent relaxation by histamine was not affected; raising the concentration of 7-NI to 10−4 M abolished the neurogenic response and partially inhibited the response to histamine, suggesting that 7-NI is a partially selective nNOS inhibitor [6]. By the use of 7-NI, Hudetz et al. [38] provided evidence suggesting that NO from a neuronal source contributes to the increase in capillary erythrocyte flow in anesthetized rats. Cerebral blood flow responses following somatosensory electrical stimulation were abolished shortly after application of 7-NI in rats [17]. 7-NI attenuated the cerebral blood flow, cerebral blood volume, and blood oxygenation level-dependent responses to electrical stimulation of the forepaw in cats, suggesting a critical role of neuronally produced NO in the cerebrovascular coupling [86]. Pial arterial diameter increased during acute hypertension, and this response was diminished by treatment with the nNOS inhibitor propyl-Larginine in anesthetized rats, supporting the hypothesis that NO released from parasympathetic nerve fibers contributes to cerebral vasodilatation during acute hypertension [88]. There is evidence suggesting that hypothermia-induced inhibition in the relaxation of canine cerebral arteries induced by electrical nerve stimulation is not associated with a decreased synthesis and release of NO in nitrergic nerves nor with a reduced ability of smooth muscle to relax in response to NO; interference with the propagation of action potentials may be involved in the inhibition via hypothermia [62]. Hypoxia-induced impairment of nitrergic nerve function is prevented by cooling, providing an idea that hypothermia plays a beneficial role in the ischemic, deteriorating brain. Tanaka et al. [89] also demonstrated that hypothermia augments porcine cerebral vasodilatation associated with nitrergic nerve activation, possibly by increasing the production of NO from L-arginine, thereby preventing impairments of NO production by hypoxia. Hypothermia itself inhibits nitrergic

Pflugers Arch - Eur J Physiol Fig. 1 Information pathways via NO liberated from nitrergic neurons to vascular smooth muscle. CaM, calmodulin; BH4, tetrahydrobiopterin; nNOS*, activated nNOS; L-Arg., Larginine; L-Citru., L-citrulline; ADMA, asymmetric dimethylarginine; SOD, superoxide dismutase; O2−, superoxide anion; Hb, oxyhemoglobin; GTP, guanosine triphosphate; GC, guanylyl cyclase; ODQ, 1H[1,2,4]oxadiazolo[4,3a]quinoxalin-1-one; cGMP, cyclic GMP; PDE-5, phosphodiesterase-5

Nitrergic Nerve impulse

nicotine

Synthetic NOS inhibitor

[ Ca2+ ] i

Ca2+

CaM BH4

L-NMMA L-NA

NOS

nNOS*

L-SMTC 7-NI

ADMA

L-Citru.

L-Arg. + O2

+ NO O2-

SOD

Hb

NO GTP

GC

ODQ

cGMP PDE-5

sildenafil

[ Ca2+ ]i Ca2+senseitivity

Relaxation

Smooth muscle

Fig. 2 Angiographical recordings of the cerebral arterial diameter before (left), during (second from left), and after (third from left) electrical stimulation of the greater petrosal nerve in the anesthetized Japanese monkey. The right figure shows a recording obtained under treatment with L-NA in the same monkey. During electrical stimulation, anterior (ACA) and middle cerebral arteries (MCA) and neighboring arterioles

dilated; after the stimulation was terminated, the vasodilator responses disappeared. Treatment with L-NA constricted the arteries and arteriole (right figure), suggesting that these vessels were dilated under control conditions following tonic release of NO from nitrergic nerves and endothelial cells. Reproduced with permission from Neuroscience 96: 393–398, 2000, Fig. 5

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nerve-mediated responses [62]. However, under hypoxic conditions, hypothermia potentiates the response to nitrergic nerve activation. Whether hypoxia therapy, increasing nitrergic nerve function, overcomes the inhibitory effect of hypothermia is unknown. Anatomical studies demonstrated that most perivascular NOS-containing neuronal elements in the rat brain corresponded to nerve terminals, and a majority of these nerves were located in the immediate vicinity of the blood vessels, suggesting that nitrergic basal forebrain neurons are involved in the flow response observed following stimulation of the basal forebrain [115]. In addition, interactions between basalocortical and intracortical NOS neurons are involved in the spatial and temporal regulation of cortical perfusion following basal forebrain activation, and they may become dysfunctional in pathologies such as Alzheimer’s disease [115]. There is evidence that tonic activity in perivascular nitrergic nerve fibers lying in close proximity to intraparenchymal microvessels may be a source of dilator tone within the hippocampus in rats [16]. In the first phase in streptozotocin-induced diabetic rats, perivascular nitrergic nerves lose their nNOS content, this phase being reversed by insulin therapy; in the second phase, nitrergic cell bodies in the ganglia are lost via apoptosis, suggesting that nitrergic degeneration in diabetic cerebral arteries appears to elucidate the link between diabetic autonomic neuropathy and stroke [18]. The rupture of aneurysm and subsequent subarachnoid hemorrhage cause fatal results. In endothelial NOS (eNOS)-deficient mice, the incidence of cerebral aneurysm was similar to that found in wild-type mice; in aneurysm walls of eNOS-deficient mice, the expression of nNOS was upregulated, compared with that in wild-type mice, suggesting the compensatory effect of nNOS [2]. Both eNOS and nNOS appear to have complementarily protective roles against aneurysm formation. Hemoglobin and hemolysate inhibit endothelium-dependent vasodilatation to ACh [53] or electrical nerve stimulation [52, 93], possibly via NO trapping and deterioration of the synthesis of cyclic guanosine monophosphate (GMP) in vascular smooth muscle [53]. Blockade of actions of NO liberated from nitrergic neurons is suggested to be a mechanism underlying cerebral vasospasm after subarachnoid hemorrhage that results in massive increase in NO-trapping substance oxyhemoglobin in subarachnoid space [93]. Acetylcholine inhibited the vasodilatation induced by nitrergic nerve stimulation in isolated canine cerebral arteries possibly through its action on prejunctional nitrergic neurons [5]. Sumatriptan, a 5-HT1B/1D/1F receptor agonist and migraine therapeutic, contracted the arteries and reduced the vasodilator response to nerve stimulation. Sumatriptan does not interfere with the release of NO from nitrergic nerves but appears to functionally antagonize NO actions by its vasoconstrictor activity. This stimulating action may minimize 5-

hydroxytryptamine (5-HT)-induced cerebral vasodilatation that is responsible for relieving migraine headache. Cilostazol, an antiplatelet agent with the type 3 phosphodiesterase inhibitory action, selectively enhanced relaxation caused by nitrergic nerve stimulation but did not affect the relaxation induced by endothelial NO in dog cerebral arterial strips [9]. This vasodilator action of cilostazol may explain the reduction in the risk of secondary stroke in addition to the antiplatelet action. Ginsenosides, the aqueous extract of the Panax ginseng, potentiated the relaxation induced by nitrergic nerve stimulation and nicotine in de-endothelialized monkey cerebral arteries that was abolished by treatment with L-NA, suggesting that ginsenoside appears to increase the synthesis or release of NO from the perivascular nerve, but the superoxide-scavenging action and phosphodiesterase inhibition are not involved in its action [96]. Acetylcholinesterase inhibitors are widely evaluated to be symptomatically effective in mild-to-moderate Alzheimer’s disease. One of the mechanisms of its action is suggested to derive from increased cerebral blood flow [31, 126]. The inhibition of acetylcholinesterase augments the action of acetylcholine released from pre-ganglionic fibers in the parasympathetic pterygopalatine ganglia and potentiates the release of NO from postganglionic nitrergic neurons, resulting in improved cerebral blood flow; this effect is expected to counteract the vicious cycle of blood flow/β-amyloid production [108]. Neurogenic acetylcholine is not involved in cerebral vasodilatation, because vasodilator responses to ganglionic stimulation are not influenced by cholinergic blocking agents in monkeys and dogs [99, 110].

Peripheral blood vessels Coronary vasculature and circulation In patients with angiographically normal coronary arteries, intracoronary infusion of L-SMTC, the nNOS-selective inhibitor, reduced basal coronary blood flow and epicardial coronary diameter but had no effect on increases in flow evoked by intracoronary-infused substance P, suggesting that nNOSderived NO regulates basal blood flow in human coronary vascular beds [79]. Tonic innervation of nitrergic nerves may be involved in coronary vasodilatation at rest; however, nNOS-derived NO liberated from other vascular and perivascular tissues cannot be excluded. In the isolated innervated rabbit heart, the vagus nerve-mediated increase in NO and the ventricular fibrillation threshold were not affected during atropine perfusion and by endothelial dysfunction, suggesting that a ventricular nitrergic parasympathetic innervation is responsible for the anti-fibrillatory action [14]. Neurogenic NO seems to act on ventricular muscle; however, whether nitrergic nerve-mediated coronary vasodilatation

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plays a beneficial role in ventricular function is not determined. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase-positive nerve fibers were found along the entire course of the accessory coronary arteries from their main branches to the arteriolar level in the rat heart [77]. Autonomic efferent axons (nitrergic, noradrenergic, and cholinergic) were more abundant around the atrial vessels than the ventricular vessels of rat heart [82]. In isolated canine coronary arteries, electrical nerve stimulation produced relaxations that were abolished by β-adrenoceptor blockers but not influenced by NOS inhibitors [85, 102]. Histological studies demonstrated the presence of NADPH diaphorase and nNOS immunoreactivity in canine and monkey coronary arteries; however, these nerves were located only in the adventitia but not in the adventitia-medial border. These findings suggest that neurogenic coronary vasodilatation is mediated mainly by adrenergic neurotransmitters. Functional roles of vasodilator nerve-derived NO in coronary vasodilatation remain to be determined. Pulmonary vasculature and circulation Electrical field stimulation in the presence of guanethidine and atropine in guinea pig pulmonary arteries denuded of the endothelium resulted in a frequency-dependent relaxation that was inhibited by treatment with L-NAME, suggesting the presence of vasodilator nitrergic neurons [78]. Nicotine, known to stimulate autonomic neurons to release transmitters including NO, induced relaxation in canine pulmonary arterial and venous strips under treatment with prazosin that was abolished by L-NA, hexamethonium, and methylene blue, suggesting that nitrergic nerves innervate pulmonary vasculatures. The nitrergic vasodilatation was larger in the artery than in the vein [8]. Electrical field stimulation-induced guinea pig pulmonary arterial relaxations were potentiated by the type 5 phosphodiesterase inhibitor zaprinast [90]. In neonatal pulmonary arteries, electrical stimulation of non-adrenergic, noncholinergic nerves resulted in relaxations that were abolished by treatment with tetrodotoxin, L-NAME, and the soluble guanylyl cyclase inhibitor 1H[1,2,4]oxadiazolo[4,3a]quinoxalin-1-one [32]. The fawn-hooded rat strain reveals a congenital predisposition to primary pulmonary hypertension; its lungs revealed a lower number of intrinsic nitrergic neurons, but these changes were not observed in the number of enteric nitrergic neurons in the esophagus, suggesting that nitrergic nerve function is selectively impaired, and this change may participate in the development of pulmonary hypertension [117]. nNOS-immunoreactive neurons innervate human pulmonary blood vessels [27]. Histolochemical and immunohistochemical studies showed that nitrergic neurons were practically restricted to large extrapulmonary arteries in guinea pigs,

whereas noradrenergic and substance P-containing axons were ubiquitous from the pulmonary trunk to smallest intraparenchymal vessels [34]. Renal vasculature and circulation Under treatment with prazosin, nicotine, an activator of autonomic neurons, induced relaxations in renal arteries from dogs and Japanese moneys; nicotine-induced relaxations were abolished by hexamethonium, L-NA, and oxyhemoglobin. Histochemical studies demonstrated perivascular nerves containing NADPH diaphorase and nNOS immunoreactivity. These findings suggest functioning of nitrergic vasodilator neurons in dog and monkey renal arteries [65]. Ichihara et al. [39] obtained evidence that the nNOS inhibitor LSMTC decreased afferent and efferent arteriolar diameters. These authors suggested that nNOS exerts a differential modulator action on the juxtamedullary microvasculature by enhancing efferent, but not afferent, arteriolar responsiveness to angiotensin II. Medullary blood flow reduced in rats treated with 7-NI, similarly to L-NAME, suggesting that NO generated by nNOS is mainly responsible for adequate perfusion of the medulla [119]. Deoxycorticosterone acetate (DOCA)-salt hypertensive rats had reduced nNOS expression in the macula densa and small afferent arteriole diameter, compared to control rats; angiotensin-converting enzyme inhibitors or thiazide increased nNOS and NO production, suggesting that afferent arteriolar diameter may be regulated by NO from nNOS in the macula densa of DOCA rats [114]. In anesthetized rabbits, treatment with L-NA increased mean arterial blood pressure and reduced renal and medullary blood flow under resting conditions and decreased the vasodilator response to renal nerve stimulation [22]. NO may protect the kidney, in particular the medulla, via vasodilatation and also hypoxia from renal nerve stimulation-induced vasoconstriction. However, whether neurogenic NO, endothelial NO, or both is involved in the response has not been determined. By the use of wildtype mice and those of eNOS null and nNOS null mutant, Mattson and Meister [54] obtained data suggesting that NO produced by nNOS mediates an increase in medullary blood flow in response to angiotensin II, whereas NO from eNOS regulates baseline blood flow in the renal cortex and medulla. In transgenic rats with inducible malignant hypertension, LSMTC increased mean arterial pressure and decreased renal plasma flow [70]. nNOS-derived NO, but yet no direct evidence of nitrergic nerve involvement, appears to exert pronounced renal vasodilatation in these rats. Upregulated nNOS gene expression in neonatal preglomerular resistance vessels in the immature pig suggests that the nNOS isoform may be responsible for counteracting angiotensin II-induced increase in vascular resistance [73]. In the Brown Norway rat that often shows exaggerated responses to NO, nNOS inhibition reduced renal blood flow and did not augment myogenic

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autoregulation, whereas iNOS inhibition reduced renal blood flow and augmented myogenic autoregulation [121]. Huang et al. [37] provided evidence showing that 7-NI impairs renal function but had no effect on blood pressure in spontaneously hypertensive rats (SHR); the nNOS/NO pathway protects against the development of kidney damage in SHR. On the other hand, Dautzenberg et al. [20] showed that the attenuating effect of NO on myogenic responses in renal blood flow autoregulation in mice depends on eNOS but not on nNOS or iNOS. Mesenteric and hepatic circulation and portal hypertension Electrical nerve stimulation or nicotine elicited relaxations in isolated dog, monkey, cow, and guinea pig mesenteric arteries treated with α-adrenoceptor antagonists that were abolished by treatment with NOS inhibitors [107]. Histochemical studies demonstrated the network of NOS-immunoreactive neurons in the adventitia and adventitio-medial border of dog and monkey mesenteric arteries [124, 125]. nNOS-immunoreactive nerves were observed in relation to hilar and interlobar vessels and to larger central veins and hepatic veins in the guinea pig liver [24]. Nitrergic nerves immunoreactive for nNOS are localized in the portal tract where nNOS colocalized with both neuropeptide P- and CGRP-containing fibers [55]. Shiraishi et al. [84] obtained evidence suggesting that the nicotine-induced relaxation is mediated by norepinephrine, CGRP, and NO released from perivascular nerves in dog hepatic arteries; the responses associated with activations of βadrenoceptors and CGRP1 receptors are predominant over those to NO. There are distinct species differences in the nitrergic innervation of mammalian liver; therefore, care must be taken in extrapolating results obtained from studies of the livers from experimental animals and humans. In SHR, dexamethazone decreased the release of neurogenic NO in response to electrical nerve stimulation, and this decrease was prevented by the glucocorticoid receptor antagonist mifepristone [3]. The expression of nNOS in mesenteric arteries from two-kidney, one-clip renovascular hypertensive rats was decreased compared with control arteries; histochemical staining of mesenteric arteries showed dense innervation of nNOS-immunopositive nerves that was less in arteries from renal hypertensive rats than that in control arteries [48]. The decrease in nNOS expression and nitrergic innervation in these arteries may lead to enhanced adrenergic neurotransmission and contribute to the initiation of renovascular hypertension. Periarterial nerve stimulation induced a dilatation in perfused mesenteric arterial vasculature of rats, which was more pronounced in portal vein-ligated rats; nNOS was localized to

the adventitia showing more intense staining and increased protein expression in portal vein-ligated rats, suggesting that perivascular nNOS-protein expression is enhanced in mesenteric arteries in portal hypertension [46]. nNOS and heat shock protein-90 expressions were increased in mesenteric nerves from portal vein-ligated rats as compared to sham rats; perivascular nerve stimulation induced mesenteric arterial dilatation to a greater extent in portal vein-ligated rats, and the stimulation-induced relaxation was markedly reduced by L-NAME and geldanamycin, a specific inhibitor of heat shock protein-90 signaling, suggesting that heat shock protein-90 acts as a signaling mediator of nNOS-dependent nerve-mediated mesenteric arterial dilatation [57]. Bile duct ligation increased nNOS messenger and protein levels in mouse aortas; hepatic artery nNOS mRNA levels in cirrhotic patients were markedly increased compared with controls, suggesting that nNOS may be involved in the regulation of systemic hyperdynamic circulation and portal hypertension [13]. The development of hyperdynamic circulation observed in shortterm portal hypertension in rats appears to be associated with decreased adrenergic influence and increased influences of nitrergic and sensory innervations [75]. Uterine vasculature and circulation Electrical or chemical (by nicotine) nerve stimulation elicited vasodilatation in guinea pig, dog, monkey, and human uterine arteries treated with α-adrenoceptor antagonists that was abolished by NOS inhibitors. Histochemical studies demonstrated the presence of nitrergic innervation in rat uterine arteries. Accumulated data on nitrergic nerve functioning and histology have been summarized in our recent review article [113]. Nitrergic nerves quite likely contribute to controlling myometrial and placental blood flow in pregnant and non-pregnant females. Whether impaired nitrergic nerve function is involved in uteroplacental blood flow reduction under pathological conditions, such as pre-eclampsia, remains to be determined. Women with high resistance placental circulation at risk of pre-eclampsia, intrauterine growth restriction, or both have raised plasma asymmetric dimethylarginine (ADMA) concentrations [76]; under such pathological conditions, vasodilator actions of NO not only from endothelia but also nitrergic nerves are impaired. Maternal undernutrition in rats enhances myogenic tone of the radial uterine arteries and reduces uteroplacental blood flow, as a result of impairment of the NO synthase pathway [118]. However, whether NO is synthesized by eNOS, nNOS, or both is not determined. Blood vessels were observed within the capsule of the major pelvic ganglia as well as outside of the capsule in rats; the extra-capsular arterioles and venules were innervated by nNOS-positive nerve fibers, suggesting that blood flow in the major pelvic ganglion may be regulated by nitrergic nerve fibers [10].

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Ocular circulation Central retinal arteries isolated from dogs and monkeys, treated with α-adrenoceptor blockers, produced relaxations in response to transmural electrical stimulation and nicotine; tetrodotoxin and hexamethonium abolished the responses to electrical stimulation and nicotine, respectively; L -NA abolished the responses to these stimuli [104, 112]. Nicotine injected into the unilateral carotid artery in anesthetized dogs induced dilatation of ipsilateral retinal arteries that was abolished by treatment with L-NA or hexamethonium [104]. Neurons containing nNOS are immunohistochemically demonstrated in monkey retinal arteries and arterioles [112]. NO produced by nNOS in nitrergic nerve terminals is suggested to be a physiological vasodilator in the rat optic nerve [83]. In the intraocular segment of the bovine long posterior ciliary artery treated with guanethidine, electrical field stimulation evoked biphasic relaxation; the first component, but not the second, was abolished by L-NAME; involvement of CGRP, substance P, and vasoactive intestinal peptide in the responses was excluded by experiments using antagonists or under desensitization to peptides [67]. NO appears to mediate the first component of neurogenic relaxation, whereas the neurotransmitter mediating the second component remains to be determined. Using histochemical and immunological methods, Bergua et al. [12] showed the presence of nNOS/NADPH-diaphorasepositive nerve fibers surrounding the posterior ciliary and central retinal arteries in rats, suggesting that these fibers are involved in ocular vasodilatation. There is evidence supporting the idea that the pterygopalatine ganglion is the source of nitrergic nerve fibers distributed to the central retinal artery and also middle and posterior cerebral arteries [101]. It was suggested that pterygopalatine and trigeminal ganglia are the most likely sources for extrinsic nitrergic nerve fibers to the human eye [33]. Nasal vasculature and circulation In human nasal mucosa, electrical nerve stimulation elicited vascular relaxations that were suppressed by treatment with L-NAME and antagonists to receptors of sensory nerve transmitters [Saptide, hCGRP-(8–37)], suggesting that neurogenic NO, substance P, and CGRP may be mediators in the non-adrenergic, non-cholinergic inhibitory nerves [66]. Relaxations induced by electrical nerve stimulation in canine nasal mucosa strips treated with atropine and guanethidine were abolished by NOS inhibitors; the responses were potentiated by lowering the bath temperature from 34 to 25 °C, whereas relaxations to sodium nitroprusside were unaffected by cold exposure [122]. It appears that cold exposure augments the NO release from nitrergic nerves; thus, enhanced

nasal vasodilatation contributes to the swelling of nasal mucosa under cold conditions. In anesthetized rats, electrical stimulation of postganglionic nerves derived from the right sphenopalatine ganglion increased nasal blood flow on the ipsilateral side and treatment with L-NA abolished the response; nNOS-containing nerve fibers were immunohistochemically demonstrated in mucosal arteries [60]. The nNOS- and NADPH-diaphorase-positive nerve fibers were found in arteries, but not veins, of the human nasal mucosa [74]. NOS-immunoreactive nerve fibers were colocalized in parasympathetic nerves in the adventitia of arterial vessels in human mucosa, suggesting that NO takes part in the regulation of physiologic processes, such as nasal blood flow [47]. Skeletal muscle vasculature and circulation Canine saphenous arteries treated with α-adrenoceptor blockers and α,β-methylene ATP responded to nicotine with relaxations that were attenuated by L-NA, suggesting a mediation of NO from autonomic efferent nerves [64]. Electrical stimulation of the superior laryngeal nerve produced hind limb vasodilatation and the fall in hindlimb vascular resistance in the rat; the responses were abolished by treatment with 7-NI, suggesting that reflex vasodilatation in the rat hind limb produced by electrical stimulation involves the release of NO from NOS-positive postganglionic lumbar sympathetic nerves [71]. Sciatic neuritive endoneural blood flow was reduced in streptozotocin-induced diabetic rats, as compared with controls; eugenol, an ingredient of clove oil having antioxidant and anti-inflammatory properties, corrected this deficit [59]. In isolated fast-twitch extensor digitorum longus muscles, but not slow-twitch soleus muscles, from mice, cyclic GMP formation increased with electrical stimulation and treatment with L-NA abolished the stimulation-induced increase in cyclic GMP; arteriolar relaxation in contracting fast-twitch muscle was attenuated in mice lacking either nNOS or eNOS, suggesting that increases in cyclic GMP and NO-dependent vasodilatation in fast-twitch skeletal muscle may require both nNOS and eNOS [49]. Brachial artery infusion of L-SMTC reduced the basal blood flow in healthy subjects but did not inhibit the acetylcholine-induced blood flow increase, suggesting that basal forearm blood flow is regulated by NO derived from nNOS [80]. Hindquarter blood flow and conductance were reduced by treatment with L-NAME in swim-trained rats to a greater extent than in sedentary rats; expression of nNOS in the hindquarter skeletal muscle was increased in swim-trained rats [91]. There was no effect of blockade of NOS on the exercise-induced increase in skeletal muscle perfusion, whereas NOS blockers reduced muscle blood flow at rest and in

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recovery from exercise [72]. However, whether NO is derived from nitrergic nerves, endothelial cells, or both has not been determined. Cutaneous vasculature and circulation In isolated canine cutaneous arteries, nicotine produced relaxations, which were not influenced by atropine but were abolished by hexamethonium: the relaxation was partially inhibited by L-NA and the remaining response was abolished by desensitization to CGRP or treatment with CGRP-(8–37), a CGRP receptor antagonist, suggesting that relaxations in response to nitrergic nerve stimulation by nicotine are mediated by NO and CGRP [116]. A similar conclusion was obtained by using nNOS inhibitors and CGRP-(8–37) in isolated rat skin microvasculature [56]. In canine isolated small labial arteries, Okamura et al. [63] obtained evidence suggesting that relaxations induced by electrical nerve stimulation are mediated exclusively by neurogenic NO, whereas nicotine evokes relaxations by a mediation of nerve-derived NO and also CGRP, possibly from sensory nerves. The effects of local changes in temperature are capable of vasoconstricting or vasodilating the skin. In humans, local warming initiates a transient vasodilatation succeeded by a plateau phase due to NO; the vasoconstriction with local skin cooling is brought about by postsynaptic upregulation of αadrenoceptors and also by inhibition of the NO system [45].

Functional balance of nitrergic and adrenergic innervation in vasculatures Summarized in Table 1 are differences of functional innervation of parasympathetic nitrergic and sympathetic adrenergic nerves in cerebral and peripheral vasculatures. In cerebral arteries, nitrergic nerve-mediated vasodilatation is evidently greater than adrenergically induced vasoconstriction; in addition, endothelium-derived NO plays an important role in counteracting vasoconstrictor interventions. Therefore, cerebral blood flow is regulated mainly by activities of nitrergic neurons and endothelial cells via the release of NO. Large cerebral arteries have substantial tone and react to vasodilator stimuli, including NO [25]. In the brain, blood flow is regulated not only by pial arterioles but also proximal arteries; therefore, arterial and arteriolar diameter changes are important factors influencing cerebral blood flow [26]; this is not the case in peripheral vasculatures, where small arteries and arterioles play a major role in the control of vascular resistance and blood flow. The NO/cyclic GMP system is expected to play pivotal roles in keeping cerebral blood flow constant and minimizing cerebral hypoxia and impaired glucose supply,

Table 1 Autonomic efferent neural control in regional vascular tone and systemic blood pressure Cerebral:

Nitrergic >> Adrenergic (α)

Coronary: Pulmonary: Renal: Mesenteric:

Adrenergic (β > α) Nitrergic? Adrenergic (α) > Nitrergic Adrenergic (α) > Nitrergic Adrenergic (α) > Nitrergic

Hepatic: Ocular: Nasal: Tongue

Adrenergic (α) > Nitrergic Adrenergic (α) > Nitrergic Adrenergic (α) > Nitrergic Adrenergic (α) + Purinergic (P 2x ) a > Nitrergic [100] Adrenergic (α) > Nitrergic

Uterine: Skeletal muscle: Cutaneous: Systemic blood pressure:

Adrenergic (α) > Nitrergic Adrenergic (α) > Nitrergic and Peptidergic b Adrenergic (α) > Nitrergic

α α-adrenergic vasoconstriction, β β-adrenergic vasodilatation, a P2x Vasoconstriction mediated via α,β-methylene ATP-sensitive receptors, b Sensory nerve-mediated vasodilatation

when acute cerebral hypoperfusion is provoked by large peripheral bleeding or hemorrhagic shock. On the other hand, peripheral circulation, except that in the heart, is regulated mainly by vasoconstricting, adrenergic nerve activity, which contributes to shift blood supply from the periphery to the brain in an emergency. Under physiological conditions, there is some buffering factor of vasodilatation via nitrergic nerves in peripheral tissues. In the periphery, the functional role of autonomic nerves innervating coronary vasculatures is quite unique: nitrergic vasodilator nerve function is unclear but adrenergically supplied norepinephrine results in coronary vasodilatation via activation of β1adrenoceptors located in arterial smooth muscle that contributes to increase coronary blood flow in cases of enforcement of cardiac muscle and increased metabolism through adrenergic activation. Cerebral blood flow and vascular resistance are mainly regulated by nitrergic neurogenic and endothelial vasodilatation, whereas peripheral vascular resistance, except that in the heart, is mainly controlled via adrenergic neurogenic vasoconstriction and coronary arterial resistance may be regulated by adrenergic neurogenic vasodilatation. Systemic treatment of deactivating adrenergic nerves in patients with cerebral ischemia decreases perfusion pressure in the brain and promotes cerebral hypoperfusion. The effective way to increase cerebral blood flow would be to increase vasodilator mediator of neurogenic and endothelial NO by reducing counteracting interventions, such as oxidative stress and endogenous NOS inhibitor ADMA. It would be quite important in early life to keep the NO system healthy by eliminating inappropriate habits of daily life (smoking, sedentary life, heavy alcohol

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intake, mental stress, high salt intake, and unbalanced diet) [95] and obesity [109]. Increasing lines of evidence suggest that vascular dysregulation via impaired endothelial and nitrergic nerve function is an early event in Alzheimer’s disease [21, 81, 108]. Cholinergic innervation was previously considered to be involved in the vasodilatation in various organs as described in Goodman and Gilman’s textbook of pharmacology [35]. However, this description has been deleted in their recent issue [123].

Systemic blood pressure Intravenous injections of L-NA raised systemic blood pressure in anesthetized dogs, and L-arginine lowered the pressure; contractions induced by electrical stimulation in isolated mesenteric arteries were potentiated by treatment with L-NA [103]. The induced hypertension is suggested to be associated partly with depletion of nNOS-derived NO. In anesthetized Japanese monkeys, intravenous injections of L-NA elevated systemic blood pressure to a significantly greater extent in monkeys under treatment with phentolamine compared to those with the ganglionic blockade, suggesting that hypertension induced by NOS inhibitors is associated with an elimination of nitrergic nerve function together with an impairment of the basal release of NO from the endothelium [61]. 7-NI increased total peripheral resistance in SHR [11]. In malignant hypertensive rats, the nNOS inhibitor L-SMTC increased mean arterial pressure to a lesser extent than that seen in normotensive rats [70]. The release of neurogenic NO under resting conditions appears to be less in the hypertensive rats. However, Huang et al. [37] reported that 7-NI impaired renal function but had no effect on blood pressure in SHR. In two-kidney, one-clip renovascular hypertensive rats, the inhibitory function of nitrergic nerves in adrenergic neurotransmission was decreased [48]. This functional alteration based on the decreased nNOS expression and nitrergic innervation leads to enhanced adrenergic neurotransmission and contributes to the initiation and development of renovascular hypertension. There is evidence showing that a functional neurogenic mechanism for increasing basilar artery blood flow or brain stem blood flow copes with increased local sympathetic activities in acutely stressful situations, and this increased blood flow as a defensive mechanism diminishes in genetic and non-genetic hypertensive rats due most likely to decreased parasympathetic nitrergic nerve activities [19]. Treatment of pre-hypertensive (4 weeks of age) SHR with the renin inhibitor aliskiren mitigates increases in plasma ADMA levels; restores L-arginine/ADMA ratios; enhances levels of nNOS-α, which was first discovered as nNOS, a 160-kDa protein; and prevents decreased nNOS-β levels in the kidney, suggesting that aliskiren is a therapeutic agent for

pre-hypertension that regulates the ADMA/NO pathway [87]. Whether nNOS is derived from nitrergic neurons or other renal tissues is not determined. In Dahl salt-sensitive hypertensive rats, treatment with ghrelin, the endogenous ligand for the growth hormone-secretagogue receptor, for 3 weeks increased urine volume, increased urine NO excretion, and tended to increase renal nNOS mRNA expression, suggesting that ghrelin counteracts salt-induced hypertension in Dahl rats through diuretic action associated with increased renal NO production, possibly through nNOS activation [1]. Renal expression of protein inhibitor of nNOS (PIN) is increased preceding hypertension, while the inhibition of PIN expression by siRNA targeting PIN attenuates the development of hypertension in SHR [120]. The authors suggest that the consideration of PIN siRNA therapy as a viable approach for restoring disturbed NO/reactive oxygen species balance, thereby preventing hypertension. Hojná et al. [36] provided evidence that reduced nNOS expression was noted in the brain stem of rats with genetic and salt-induced hypertension, and this was associated with increased blood pressure due to enhanced sympathetic tone. Impairment of perivascular nitrergic nerve functions reduces the ability of NO to counteract sympathetic vasoconstriction, resulting in increased vascular resistance [98]. Neurogenic and endothelial NO acting on neurons in the medulla and hypothalamus appears to interfere with central sympathetic nerve activity, leading to a decrease in adrenergic influences on resistance vessels and, as a result, systemic blood pressure decrease.

Nitrergic neurogenic vasodilatation in non-mammals Functional role of nitrergic nerves In ring preparations of the aortic anastomosis in the estuarine crocodile, the addition of L-NAME increased the basal tone; adrenaline-induced vasoconstrictions were counteracted by reflex vasodilatations that were susceptible to blockade by LNAME; immunohistochemical study revealed the presence of NO synthase, suggesting that a sphincter-like function for this vessel is controlled by the nitrergic mechanism [4]. Nicotine induced vasodilatation of the dorsal aorta and the intestinal vein of the short-finned eel, which was not influenced by removal of the endothelium but was blocked by L-NA and the nNOS inhibitor NG-propyl-L-arginine, suggesting that neurally derived NO contributes to the maintenance of vascular tone in this species [41]. In large veins of the toad, Bufo marinus, the nNOS inhibitor vinyl-L-NIO reduced acetylcholine-induced vasodilatation in the presence of phenoxybenzamine, which was endothelium-independent; nNOS-immunoreactive nerves were present [15]. Vasodilatation induced by nicotine in

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pulmocutaneous vasculature of the toad was inhibited by 1H-[1,2,4]oxalodiazolol[4,3a]quinoxalin-1-one, L NA, and vinyl-L-NIO but was resistant to the removal of the endothelium, suggesting that NO released from nitrergic nerves but not from the endothelium regulates the toad arteries [42]. The nitrergic vasodilatation appears to be caused, in part, via activation of ATPsensitive K+ channels. Histochemical studies There are nNOS-immunoreactive nerve fibers located in the hepatic artery, portal vein, and biliary duct that form the hepatic hilum in the rainbow trout [23]. The immunoreactive nitrergic nerve plexus reaches the kidney along the vasculature, mainly running with the postcardinal vein in the rainbow trout; the collecting tubules and ducts, large arteries, and glomerular arterioles of the tubular middle and posterior trunks were innervated by nitrergic nerves, suggesting that nitrergic neural structures may be involved in the control of renal functions [44]. There is nitrergic innervation in the large pulmonary veins in two frog species [127] and the dorsal aorta and intestinal vein of the eel [41]. The nNOS-positive neurons are suggested to mediate vasodilatation via the release of NO.

Summary Recent advances in research on nitrergic nerve-mediated vasodilatation and blood flow increase in cerebral and peripheral vasculatures and on the systemic blood pressure regulation via NO liberated from perivascular nerves are summarized in this review. Nitrergic neurogenic vasodilatation is evidently recognized in cerebral vasculatures, because counteracting vasoconstrictor actions via adrenergic nerve activation are quite weak. Nitrergic neurons, as parasympathetic postganglionic nerves, innervating cerebral arteries and arterioles receive impulses from the superior salivatory nucleus through the greater petrosal nerve. Postganglionic cholinergic nerves, although histologically determined, do not play a role as dilators in the cerebral vasculature, because cholinergic blockers are ineffective in inhibiting cerebral vasodilatation induced by stimulation of postganglionic neurons. Improvement of vasodilatation and blood flow increase via the NO/cyclic GMP pathway in the brain is an important target for prophylaxis and treatment against ischemic brain diseases and Alzheimer’s disease. Evident vasoconstrictions are elicited in response to postganglionic nerve stimulation in vasculatures, other than cerebral and coronary arteries; only when adrenergic vasoconstrictor responses are suppressed by α-adrenoceptor antagonists or adrenergic neuron blockers, NO-mediated, neurogenic vasodilatation is recognizable. In cutaneous vasculatures,

polypeptides, such as CGRP possibly derived from sensory nerves, also participate in vasodilatation. In coronary arteries, vasodilatation induced by nerve stimulation is mainly mediated by norepinephrine via an activation of β1-adrenoceptors; evidence for neurogenic NO-mediated vasodilatation remains to be determined. In humans, coronary vasodilatation is impaired by treatment with a selective nNOS inhibitor; however, whether liberated NO is derived from nitrergic neurons or other tissues in and around coronary arteries has not been determined. The nNOS-derived NO from tissues other than nitrergic nerves was evidenced in the kidney. Table 1 summarizes the role of nerve-derived NO and norepinephrine in various mammalian vasculatures. There are regional differences in balancing of nitrergic and adrenergic nerve functions between cerebral vs. peripheral arteries and also coronary vs. other arteries. However, there is no doubt about the physiological roles of nitrergic vasodilator nerves in the control of vascular tone, vascular resistance, blood flow, and blood pressure, although roles of nitrergic nerves in coronary vasculature are yet clarified. Acetylcholine liberated from postganglionic neurons may interfere with NO release from nitrergic nerves [97]. It was proposed in our previous article [104] that functional roles of autonomic postganglionic innervation in vasculatures will be rewritten as adrenergic vasoconstrictor (or dilator) nerves and nitrergic vasodilator nerves in cerebral and peripheral vasculatures, respectively. We would like to emphasize again that there is dual autonomic innervation (adrenergic and nitrergic) present in vasculatures, in contrast to the reciprocal adrenergic and cholinergic innervation in extravascular organs and tissues, widely accepted for many years. The nitrergic nerve/cyclic GMP vasodilatation system may already be established in phylogenically old non-mammals. Acknowledgments This work was supported in part by grant-in-aid for scientific research from the Ministry of Education, Culture, Sport, Science and Technology of Japan (23390055). Conflict of interest The authors have no conflict of interest in any matter related to this work.

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