Examining multiple sleep behaviors and diurnal salivary cortisol and alpha-amylase: Within- and between-person associations.

Sleep has been linked to the daily patterns of stress-responsive physiological systems, specifically the hypothalamic-pituitary-adrenal (HPA) axis and autonomic nervous system (ANS). However, extant research examining sleep and diurnal patterns of cortisol, the primary end product of the HPA axis, has primarily focused on sleep duration with limited attention on other facets of sleep. For example, it is not clear how specific aspects of sleep (e.g., sleep quality, sleep duration variability) are related to specific components of diurnal cortisol rhythms. Salivary alpha-amylase (sAA) has been recognized as a surrogate marker of ANS activity, but limited research has explored relations between sleep and sAA diurnal rhythms. The current study utilized an ecological momentary assessment protocol to examine within- and between-person relations between several facets of sleep behavior using multiple methods (e.g., subjective report, actigraphy) and salivary cortisol and sAA. Older adolescents (N=76) provided saliva samples and diary entries five times per day over the course of three days. Sleep was assessed via questionnaire, through daily diaries, and monitored objectively using actigraphy over a four day period. Between-person results revealed that shorter average objective sleep duration and greater sleep duration variability were related to lower levels of waking cortisol and flatter diurnal slopes across the day. Within-person results revealed that on nights when individuals slept for shorter durations than usual they also had lower levels of waking cortisol the next day. Sleep was not related to the cortisol awakening response (CAR) or diurnal patterns of sAA, in either between-person or within-person analyses. However, typical sleep behaviors measured via questionnaire were related to waking levels of sAA. Overall, this study provides a greater understanding of how multiple components of sleep, measured in naturalistic environments, are related to cortisol and sAA diurnal rhythms, and how day-to-day, within-person changes in sleep duration contribute to daily variations in cortisol.

Salivary alpha amylase levels in youths with anxiety disorders.

It is suggested that salivary alpha-amylase (sAA) may be a marker of sympathoadrenal medullary system activity. Thus, it can be a possible relationship sAA and anxiety disorders. The aim of this study is to investigate sAA in children and adolescents with anxiety disorders and healthy controls. Thirty drug-free youths, aged 8-16 years, who were diagnosed as any anxiety disorders and 36 healthy controls with similar socio-demographic characteristics were included in this study. The sAA was found to be significantly increased in anxiety group compared to control group. However, there was no correlation between sAA and any anxiety scores of the scales. Present study suggested that anxiety disorders in youths may be associated with increased autonomic activity.

Impact of ectonucleotidases in autonomic nervous functions.

Adenine and uracil nucleotides play key functions in the autonomic nervous system (ANS). For instance, ATP acts as a neurotransmitter, co-transmitter and neuromodulator in the ANS. The purinergic system encompasses (1) receptors that respond to extracellular purines, which are designated as P1 and P2 purinoceptors, (2) purine release and uptake, and (3) a cascade of enzymes that regulate the concentration of purines near the cell surface. Ectonucleotidases and adenosine deaminase (ADA) are enzymes responsible for the hydrolysis of ATP (and other nucleotides such as ADP, UTP, UDP, AMP) and adenosine, respectively. Accordingly, these enzymes are expected to play an important role in the control of neuro-effector transmission in tissues innervated by both the sympathetic and parasympathetic divisions of the ANS. Indeed, ectonucleotidases have the ability to either terminate P2 receptor responses initiated by nucleoside triphosphates (ATP and UTP), and/or to favor the activation of ADP (e.g. P2Y1,12,13) and UDP (e.g. P2Y6) and/or adenosine (P1) specific receptors. In addition, ectonucleotidases can also importantly protect some P2 receptors from desensitization (e.g. P2X1, P2Y1). In this review, we present the (putative) roles of ectonucleotidases and ADA in the ANS with a focus on their regulatory activity at neuro-effector junctions in the following tissues: heart, vas deferens, urinary bladder, salivary glands, blood vessels and the intestine. We also present their implication in nociceptive transmission.

Determinants of salivary evening α-amylase in a large sample free of psychopathology.

OBJECTIVE: Recently, salivary alpha-amylase (sAA) has been proposed as a suitable index for sympathetic activity and dysregulation of the autonomic nervous system (ANS). Although determinants of sAA have been described, they have not been studied within the same study with a large sample size without potential disturbances of psychopathology. In this paper, we report about correlates of evening sAA in saliva. METHODS: In 487 participants (mean age=42.9years, 59.8% female) without lifetime psychiatric disorders from the Netherlands Study of Depression and Anxiety (NESDA), sociodemographic, health and sampling determinants of sAA levels were examined using multivariable linear regression analysis. sAA was measured in two saliva samples that participants collected in the late evening, at 22:00h and 23:00h, after which these were averaged. RESULTS: In multivariate analysis, age (ß=0.20, p<0.001) and daily alcohol intake (ß=-0.13, p=0.01) were independent determinants of evening sAA levels. Gender, allergy or lung disease, and the use of oral contraceptives were univariate correlates, but no longer associated with sAA in the multivariate model. CONCLUSIONS: Age and alcohol use were identified as potential confounding factors that should be taken into account in epidemiologic studies that examine the ANS function using sAA.

PVN adenovirus-siRNA injections silencing either NOX2 or NOX4 attenuate aldosterone/NaCl-induced hypertension in mice.

Mineralocorticoid excess increases superoxide production by activating NADPH oxidase (NOX), and intracerebroventricular infusions of NADPH oxidase inhibitors attenuate aldosterone (Aldo)/salt-induced hypertension. It has been hypothesized that increased reactive oxygen species (ROS) in the brain may be a key mechanism in the development of hypertension. The present study investigated the brain regional specificity of NADPH oxidase and the role of NOX2 and NOX4 NADPH oxidase subunits in the hypothalamic paraventricular nucleus (PVN) in Aldo/salt-induced hypertension. PVN injections of adenoviral vectors expressing small interfering (si)RNA targeting NOX2 (AdsiRNA-NOX2) or NOX4 (AdsiRNA-NOX4) mRNAs were used to knock down NOX2 and NOX4 proteins. Three days later, delivery of Aldo (0.2 mg·kg(-1)·day(-1) sc) via osmotic pump commenced and 1% NaCl was provided in place of water. PVN injections of either AdsiRNA-NOX2 or AdsiRNA-NOX4 significantly attenuated the development of Aldo/NaCl-induced hypertension. In an additional study, Aldo/salt-induced hypertension was also significantly attenuated in NOX2 (genomic) knockout mice compared with wild-type controls. When animals from both functional studies underwent ganglionic blockade, there was a reduced fall in blood pressure in the NOX2 and NOX4 knockdown/knockout mice. Western blot analyses of the PVN of siRNA-NOX2- or siRNA-NOX4-injected mice confirmed a marked reduction in the expression of NOX2 or NOX4 protein. In cultured PVN neurons, silencing either NOX2 or NOX4 protein production by culturing PVN cells with siRNA-NOX2 or siRNA-NOX4 attenuated Aldo-induced ROS. These data indicate that both NOX2 and NOX4 in the PVN contribute to elevated sympathetic activity and the hypertensivogenic actions induced by mineralocorticoid excess.

Tetrahydrobiopterin: pleiotropic roles in cardiovascular pathophysiology.

Tetrahydrobiopterin (BH4) functions as a cofactor in several important enzyme systems. Substantial evidence implicates BH4 as a key regulator of endothelial nitric oxide synthase (eNOS) in the setting of endothelial dysfunction and atherosclerosis. Investigators have now taken early steps in addressing the potential of BH4 as a therapeutic strategy. However, it has become more apparent that the role of BH4 in other enzymatic pathways, including other NOS isoforms and the aromatic amino acid hydroxylases, may have a bearing on important aspects of cardiovascular homeostasis. Together with eNOS, these enzymes may play key roles in diverse cardiovascular disease states such as ischaemia-reperfusion injury, cardiac hypertrophy, cardiac autonomic function and pulmonary hypertension. This review provides an overview of the role of BH4 in cardiovascular pathophysiology.

Human tyrosine hydroxylase natural allelic variation: influence on autonomic function and hypertension.

The catecholamine biosynthetic pathway consists of several enzymatic steps in series, beginning with the amino acids phenylalanine and tyrosine, and eventuating in the catecholamines norepinephrine (noradrenaline) and epinephrine (adrenaline). Since the enzyme tyrosine hydroxylase (TH; tyrosine 3-mono-oxygenase; EC 1.14.16.2; chromosome 11p15.5) is generally considered to be rate-limiting in this pathway, probed as to whether common genetic variation at the TH gene occurred, and whether such variants contributed to inter-individual alterations in autonomic function, either biochemical or physiological. We began with sequencing a tetranucleotide (TCAT) repeat in the first intron, and found that the two most common versions, (TCAT)(6) and (TCAT)(10i), predicted heritable autonomic traits in twin pairs. We then conducted systematic polymorphism discovery across the ~8 kbp locus, and discovered numerous variants, principally non-coding. The proximal promoter block contained four common variants, and its haplotypes and SNPs (especially C-824T, rs10770141) predicted catecholamine secretion, environmental stress-induced BP increments, and hypertension. Finally, we found that two of the common promoter variants, C-824T (rs10770141) and A-581G (rs10770140), were functional in that they differentially affected transcriptional activity of the isolated promoter, disrupted recognition motifs for specific transcription factor binding, altered the promoter responses to the co-transfected (exogenous) factors, and bound the endogenous factors in the chromatin fraction of the nucleus. We concluded that common variation in the proximal TH promoter is functional, giving rise to changes in autonomic function and consequently cardiovascular risk.

Angiotensin-converting enzyme 2: central regulator for cardiovascular function.

Angiotensin-converting enzyme 2 (ACE2) is a new component of the renin-angiotensin system (RAS). Accumulating evidence shows that ACE2 provides protective effects in peripheral tissues and has great potential for the treatment of RAS-related diseases. The role of ACE2 in the central nervous system is not well established. However, in recent years, much more progress has been made on the studies of this carboxypeptidase in the central regulation of blood pressure and cardiovascular function in general. It has been shown that brain ACE2 interacts with the other components of the RAS (ACE, angiotensin II, and angiotensin II type 1 receptor), protects baroreflex and autonomic function, stimulates nitric oxide release, reduces oxidative stress, and prevents the development of or attenuates hypertension. These data support the critical role of ACE2 in the central regulation of cardiovascular function. This review summarizes recently published data on the central effects of ACE2 in the regulation of cardiovascular function.

Chronic myocardial infarction induces phenotypic and functional remodeling in the guinea pig cardiac plexus.

Chronic myocardial infarction (CMI) is associated with remodeling of the ventricle and evokes adaption in the cardiac neurohumoral control systems. To evaluate the remodeling of the intrinsic cardiac nervous system following myocardial infarction, the dorsal descending coronary artery was ligated in the guinea pig heart and the animals were allowed to recover for 7-9 wk. Thereafter, atrial neurons of the intrinsic cardiac plexus were isolated for electrophysiological and immunohistochemical analyses. Intracellular voltage recordings from intrinsic cardiac neurons demonstrated no significant changes in passive membrane properties or action potential configuration compared with age-matched controls and sham-operated animals. The intrinsic cardiac neurons from chronic infarcted hearts did demonstrate an increase in evoked action potential (AP) frequency (as determined by the number of APs produced with depolarizing stimuli) and an increase in responses to exogenously applied histamine compared with sham and age-matched controls. Conversely, pituitary adenylate cyclase-activating polypeptide (PACAP)-induced increases in intrinsic cardiac neuron-evoked AP frequency were similar between control and CMI animals. Immunohistochemical analysis demonstrated a threefold increase in percentage of neurons immunoreactive for neuronal nitric oxide synthase (NOS) in CMI animals compared with control and the additional expression of inducible NOS by some neurons, which was not evident in control animals. Finally, the density of mast cells within the intrinsic cardiac plexus was increased threefold in preparations from CMI animals. These results indicate that CMI induces a differential remodeling of intrinsic cardiac neurons and functional upregulation of neuronal responsiveness to specific neuromodulators.

Dopamine beta-hydroxylase deficiency.

Dopamine beta-hydroxylase (DbetaH) deficiency is a very rare form of primary autonomic failure characterized by a complete absence of noradrenaline and adrenaline in plasma together with increased dopamine plasma levels. The prevalence of DbetaH deficiency is unknown. Only a limited number of cases with this disease have been reported. DbetaH deficiency is mainly characterized by cardiovascular disorders and severe orthostatic hypotension. First symptoms often start during a complicated perinatal period with hypotension, muscle hypotonia, hypothermia and hypoglycemia. Children with DbetaH deficiency exhibit reduced ability to exercise because of blood pressure inadaptation with exertion and syncope. Symptoms usually worsen progressively during late adolescence and early adulthood with severe orthostatic hypotension, eyelid ptosis, nasal stuffiness and sexual disorders. Limitation in standing tolerance, limited ability to exercise and traumatic morbidity related to falls and syncope may represent later evolution. The syndrome is caused by heterogeneous molecular alterations of the DBH gene and is inherited in an autosomal recessive manner. Restoration of plasma noradrenaline to the normal range can be achieved by therapy with the synthetic precursor of noradrenaline, L-threo-dihydroxyphenylserine (DOPS). Oral administration of 100 to 500 mg DOPS, twice or three times daily, increases blood pressure and reverses the orthostatic intolerance.

Cardiac neurobiology of nitric oxide synthases.

Nitric oxide (NO) is a potent modulator of cardiac and vascular regulation. Its role in cardiac-autonomic neural signaling has received much attention over the last decade because of the ability of NO to alter cardiac sympathovagal balance to favor more anti-arrhythmic states. Complexity and controversy have arisen, however, because of the numerous sources of NO in the brain, peripheral nerves, and cardiomyocytes, all of which are potential regulators of cardiac excitability and calcium signaling. This review addresses the integrative role of NO as a relatively ubiquitous signaling molecule with respect to cardiac neurobiology. The present idea, that divergent NO-signaling pathways from multiple sources within the heart and nervous system converge to modulate cardiac excitability and impact on morbidity and mortality in health and disease, is discussed.

Functional allelic heterogeneity and pleiotropy of a repeat polymorphism in tyrosine hydroxylase: prediction of catecholamines and response to stress in twins.

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, has a common tetranucleotide repeat polymorphism, (TCAT)(n). We asked whether variation at (TCAT)(n) may influence the autonomic nervous system and its response to environmental stress. To understand the role of heredity in such traits, we turned to a human twin study design. Both biochemical and physiological autonomic traits displayed substantial heritability (h(2)), up to h(2) = 56.8 +/- 7.5% (P < 0.0001) for norepinephrine secretion, and h(2) = 61 +/- 6% (P < 0.001) for heart rate. Common (TCAT)(n) alleles, particularly (TCAT)(6) and (TCAT)(10i), predicted such traits (including catecholamine secretion, as well as basal and poststress heart rate) in allele copy number dose-dependent fashion, although in directionally opposite ways, indicating functional allelic heterogeneity. (TCAT)(n) diploid genotypes (e.g., [TCAT](6)/[TCAT](10i)) predicted the same physiological traits but with increased explanatory power for trait variation (in contrast to allele copy number). Multivariate ANOVA documented genetic pleiotropy: joint effects of the (TCAT)(10i) allele on both biochemical (norepinephrine) and physiological (heart rate) traits. (TCAT)(6) allele frequencies were lower in normotensive twins at genetic risk of hypertension, consistent with an effect to protect against later development of hypertension, and suggesting that the traits predicted by these variants in still-normotensive subjects are early, heritable, "intermediate phenotypes" in the pathogenetic scheme for later development of sustained hypertension. We conclude that common allelic variation within the tyrosine hydroxylase locus exerts a powerful, heritable effect on autonomic control of the circulation and that such variation may have implications in later development of cardiovascular disease traits such as hypertension.

Immunohistochemical investigations of the autonomous innervation of the cervine testis.

The innervation of the cervine testis was studied in 6 roe deers, 7 red deers and 14 fallow deers. The results for the three species are rather similar. With anti-sera to neurofilament (NF) and neuron specific enolase (NSE), all small and large nerve fascicles can be demonstrated, but single fibers are incompletely stained. Immunoreactions against protein gene product-9.5 (PGP-9.5) and GAP-43 (growth-associated protein-43) are better suited to depict the complete innervation pattern. Bundles of the superior spermatic and inferior spermatic nerves reach the testis via three access routes as funicular, mesorchial and caudal nerve contributions. We found no morphological evidence that the nerves in the cervine testis are directly involved in regulating Leydig cell function or seminiferous tubular motility. The majority of the testicular nerves are associated with the testicular arteries, but the musculature in the walls of the venous plexus pampiniformis is also innervated. All vascular nerve fibers represent postjunctional sympathetic axons displaying a strong dopamine-beta-hydroxylase (DBH) activity, mostly co-expressed with neuropeptide Y (NPY). The presence of cholinergic fibers in the testis of the deer is only sporadic and probably of no functional importance. In all three species of deer, a small quantity of myelinated nerve fibers is encountered in spermatic cord and tunica albuginea and regarded as afferent. The viscerosensory quality in the testicular intrinsic innervation is very likely mediated by the CGRP (calcitonin gene-related peptide)-positive fibers that run independently from the testicular vessels and end in the connective tissue of spermatic cord and tunica albuginea. The testis of the red deer contains significantly more VIP (vasoactive intestinal polypeptide)-positive axons than that of roe and fallow deer. The nerve density in the interior of the testicular lobules shows no regional differences, but there are age- and season-related changes that correlate with the developmental and functional state of the seminiferous tubules. Small testes with solid and narrow tubules, as seen in the prepuberal phase and during seasonal reproductive quiescence, are better innervated than large testes with expanded and spermatogenetically active seminiferous tubules.

Segment-specific decrease of both catecholamine concentration and acetylcholinesterase activity are accompanied by nerve refinement in the rat cauda epididymis during sexual maturation.

In the present work, histochemical and biochemical studies were conducted to analyze changes in the pattern of autonomic innervation during sexual maturation, using the rat epididymis as a model. Glyoxylic acid histochemistry and immunohistochemical studies against dopamine beta-hydroxylase (DbetaH) and acetylcholinesterase (AChE) indicated a reduction in the amount of catecholaminergic and AChE-positive neurons, fibers, and puncta detected in the cauda epididymis of adult rats (120 days old), when compared to immature (40 days) and young adult (60 days) animals. No obvious age-related variations were detected in the few catecholaminergic and AChE-positive fibers and puncta present in the caput region. AChE-positive fibers were found sorting out among epithelial cells and ending free upon the epithelial surface or into the tubular lumen of the cauda region of adult rats. Furthermore, a positive staining for AChE in epithelial cells was also detected in the caput and cauda epididymis in all ages studied. Biochemical analysis confirmed a significant decrease in noradrenaline concentration as well as AChE activity in the cauda epididymis with sexual maturation. Immunohistochemical studies against microtubule-associated protein 1B (MAP 1B), a neuronal cytoskeletal marker, further substantiated the quantitative changes observed in catecholaminergic and AChE-positive neuronal elements in the cauda epididymis. Thus, our results documented segment-specific variations in noradrenaline concentration and AChE activity during epididymal sexual maturation and suggest that such variations result, at least in part, from the refinement of the autonomic innervation pattern with age.

G(z alpha) deficient mice: enzyme levels in the autonomic nervous system, neuronal survival and effect of genetic background.

Our laboratory has generated a genetically mutant mouse in which the alpha subunit of the heterotrimeric GTP binding protein, G(z) has been made dysfunctional by homologous recombination to determine its in vivo function. These animals show a characteristic failure to thrive phenotype. G(z alpha) is expressed in a variety of nervous system tissues as well as in the adrenal medulla. We therefore examined the autonomic nervous system of the G(z alpha) deficient mouse by measuring the activity of tyrosine hydroxylase and choline acetyltransferase in the superior cervical ganglia, submaxillary gland and the adrenal medulla. Preliminary results using animals of mixed BALB/c and C57BL/6 strains gave inconsistent results. Further experiments demonstrated differences in the activity of tyrosine hydroxylase and choline acetyltransferase between BALB/c and C57BL/6 mouse strains. The analysis of the pure strains showed a reduction in the size and enzyme levels of the adrenal gland and submaxillary glands of the G(z alpha) deficient mouse suggesting a role for adrenal insufficiency and/or nutritional disorders for the failure to thrive phenotype. The survival of sympathetic and sensory neurons was also examined in the G(z alpha) deficient mouse and in the presence of pertussis toxin, sympathetic but not sensory neuronal survival in G(z alpha) deficient mice was significantly attenuated. This suggests that in vivo other pertussis toxin sensitive G proteins may be recruited to compensate for the loss of G(z alpha).

Neuronal nitric oxide synthase and the autonomic innervation of the mouse lacrimal gland.

PURPOSE: To determine the expression patterns of the vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH) and neuronal nitric oxide synthase (nNOS) in the pterygopalatine ganglion (PPG) and the exorbital lacrimal gland of normal mice. METHODS: Mouse PPG and lacrimal glands were processed for single- and double-labeled indirect immunofluorescence studies. Slides were examined with conventional fluorescence microscopy and confocal laser scanning microscopy. RESULTS: All the somata in the PPG expressed both VAChT and nNOS immunoreactivity (IR). The postganglionic axons within the ganglion showed less VAChT-immunoreactive intensity than that seen in the somata, whereas nNOS IR was almost undetectable. In the lacrimal gland, nNOS-positive nerve bundles and fibers were observed to be associated with tear-collecting ducts, blood vessels, and acini. Some nNOS-positive punctate elements appeared to be distributed among acini. Many nerve fibers were VAChT immunoreactive and a small number of fibers were TH immunoreactive in the gland. Most of the VAChT-positive fibers and some of the TH-positive nerves displayed nNOS IR. CONCLUSIONS: The expression of nNOS in cells of the PPG and in lacrimal gland nerves suggests that NO may play a role in modulating tear production. The site of action may include the PPG, ducts, blood vessels, acini, nerve fibers, and myoepithelial cells within the gland. NO may modulate parasympathetic and/or sympathetic synaptic transmission or by acting directly on lacrimal gland components. The interaction between NO-ergic and the conventional autonomic input illustrates the complexity of the innervation pattern of the mouse lacrimal gland.

The distribution and colocalisation of nicotinamide adenine dinucleotide phosphate reduced-diaphorase (NADPH-d)-/nitric oxide synthase (NOS)-containing neurons in the innervation of the duck ureter.

ensp;The distribution and colocalisation of nicotinamide adenine dinucleotide phosphate reduced-diaphorase (NADPH-d)-/nitric oxide synthase (NOS)-containing (nitrergic) neurons in the innervation of the duck ureter have been studied using histochemistry and immunohistochemistry. Quantitative analysis showed that nitrergic neurons made up 60% and 70% of the total intramural and adventitial neuronal populations, respectively. About 40% of intramural nitrergic neurons expressed VIP-immunoreactivity, and about 75% of nitrergic adventitial neurons expressed TH-immunoreactivity. The density of nitrergic adventitial neurons was significantly greater in the lower tract than in the upper and intermediate tracts. Nerve lesioning experiments showed that the majority of ureteral nitrergic innervation was extrinsic in origin; nitrergic adventitial neurons primarily projected caudocranially, whereas NOS-immunoreactive and NOS-/VIP-immunoreactive intramural neurons primarily projected craniocaudally. These findings suggest that, in birds, the nitrergic innervation plays a role in ureteral functions such as epithelial mucosecretion, muscular motility, and the closing and/or opening of the ureteral papilla.

Comparative localization of heme oxygenase-2 and nitric oxide synthase in the autonomic innervation to the human ductus deferens and seminal vesicle.

PURPOSE: The aim of present study was to determine the topographic relationship between heme oxygenase-2 (HO-2), which synthesizes carbon monoxide (CO), and neuronal nitric oxide synthase (nNOS), which generates nitric oxide (NO), in the autonomic nerves of the human ductus deferens and seminal vesicle. MATERIALS AND METHODS: Specimens of the ductus deferens and seminal vesicle were obtained during cancer surgery or vasectomy. HO-2 and nNOS were localized by indirect immunofluorescence. Additionally, the histochemical NADPH-diaphorase (NADPH-d) activity of NOS was demonstrated using a standard staining method and some modifications. RESULTS: Anti-HO-2 labeling stained virtually all nerve cell bodies in local ganglia of the pelvic plexus, which is composed of a mixed population of postganglionic sympathetic and parasympathetic neurons supplying the pelvic viscera. Furthermore, nerve cell bodies in the wall of the seminal vesicle, which are considered an extension of the pelvic plexus, were also found to stain positively for HO-2. Some of the HO-2-immunoreactive ganglion cells were also nNOS-positive, their proportion varying between individual ganglia but generally not exceeding 20%. Both enzymes were present in large adventitial nerve trunks. Only nNOS but no HO-2 was found in small intramuscular and mucosal nerve fibers. In both the ductus deferens and seminal vesicle, the highest density of nNOS-containing nerve fibers was in the lamina propria of the mucosa. A well-developed plexus of nNOS-positive nerve fibers was also observed in the muscular layer of the seminal vesicle. By contrast, there was a very sparse innervation by nNOS-positive nerve fibers in the muscle coat of the ductus deferens. In addition, a population of epithelial cells in the seminal vesicle may contain an isoform of NOS, as revealed by a resistant NADPH-d activity. CONCLUSIONS: These findings set the scene for functional studies which will hopefully clarify the biological role of CO and NO in the control of the ductus deferens and seminal vesicle.

Extrinsic and intrinsic cholinergic systems of the vascular wall.

Immunohistochemical, biochemical and functional studies have revealed two separate cholinergic systems in the arterial vascular wall. Endothelial cells represent the ubiquitous intrinsic, intimal system; they contain the acetylcholine-synthesizing enzyme, choline acetyltransferase, release a choline ester, and contain functional muscarinic receptors. Perivascular autonomic nerve fibres represent the extrinsic, adventitial system. These axons are not ubiquitous but show a highly selective distribution among and even within organs, and utilize co-mediators (NO, neuropeptides) in an organ-specific pattern. We put forward the hypothesis that the intrinsic, intimal system serves as a general regulator of basal vascular tone and wall structure responding to local, luminal stimuli, whereas the perivascular nerve fibres act on top of this basal tone by providing fine tuning in response to reflex activation due to systemic demands.