Basal and Stress-Induced Network Activity in the Adrenal Medulla In Vivo.

The adrenal medulla plays a critical role in mammalian homeostasis and the stress response. It is populated by clustered chromaffin cells that secrete epinephrine or norepinephrine along with peptides into the bloodstream affecting distant target organs. Despite been heavily studied, the central control of adrenal medulla and in-situ spatiotemporal responsiveness remains poorly understood. For this work, we continuously monitored the electrical activity of individual adrenomedullary chromaffin cells in the living anesthetized rat using multielectrode arrays. We measured the chromaffin cell activity under basal and physiological stress conditions and characterized the functional micro-architecture of the adrenal medulla. Under basal conditions, chromaffin cells fired action potentials with frequencies between ~0.2 and 4 Hz. Activity was almost completely driven by sympathetic inputs coming through the splanchnic nerve. Chromaffin cells were organized into independent local networks in which cells fired in a specific order, with latencies from hundreds of microseconds to a few milliseconds. Electrical stimulation of the splanchnic nerve evoked almost exactly the same spatiotemporal firing patterns that occurred spontaneously. Hypoglycemic stress, induced by insulin administration resulted in increased activity of a subset of the chromaffin cells. In contrast, respiratory arrest induced by lethal anesthesia resulted in an increase in the activity of virtually all chromaffin cells before cessation of all activity. These results suggest a stressor-specific activation of adrenomedullary chromaffin cell networks and revealed a surprisingly complex electrical organization that likely reflects the dynamic nature of the adrenal medulla's neuroendocrine output during basal conditions and during different types of physiological stress.

Stress system dysfunction revealed by integrating reactivity of stress pathways to psychological stress in lean and overweight/obese men.

Although the patterns of response within the sympathoadrenal medullary (SAM) system and hypothalamo-pituitary adrenal (HPA) axis are interesting and important in their own accord, the overall response to acute psychological stress involves reactivity of both pathways. We tested the hypothesis that consideration of the integrated response of these pathways may reveal dysregulation of the stress systems, which is not evident when considering either system alone. Age-matched lean and overweight/obese men were subjected to a Trier Social Stress Test and reactivity of the SAM system (salivary α-amylase, systolic blood pressure, diastolic blood pressure, and heart rate) and the HPA axis (salivary cortisol) were measured. Relative reactivity of SAM system and HPA axis was calculated as the ratio between the measures from each pathway. Although analysis of reactivity of individual stress pathways showed no evidence of dysfunction in overweight/obese compared with lean men, analysis of HPA/SAM reactivity revealed significantly lower cortisol over systolic blood pressure (CoSBP) and cortisol over diastolic blood pressure (CoDBP) reactivity in overweight/obese compared with lean men. Other measures of HPA/SAM reactivity and all measures of SAM/HPA reactivity were unaltered in overweight/obese compared with lean men. These findings suggest that the cortisol response per unit of blood pressure response is blunted in men with elevated adiposity. Furthermore, these findings support a notion of a coordinated overall approach to activation of the stress pathways with the degree of activation in one pathway being related to the degree of activation in the other.

HIF-1α is required for development of the sympathetic nervous system.

The molecular mechanisms regulating sympathetic innervation of the heart during embryogenesis and its importance for cardiac development and function remain to be fully elucidated. We generated mice in which conditional knockout (CKO) of the Hif1a gene encoding the transcription factor hypoxia-inducible factor 1α (HIF-1α) is mediated by an Islet1-Cre transgene expressed in the cardiac outflow tract, right ventricle and atrium, pharyngeal mesoderm, peripheral neurons, and hindlimbs. These Hif1aCKO mice demonstrate significantly decreased perinatal survival and impaired left ventricular function. The absence of HIF-1α impaired the survival and proliferation of preganglionic and postganglionic neurons of the sympathetic system, respectively. These defects resulted in hypoplasia of the sympathetic ganglion chain and decreased sympathetic innervation of the Hif1aCKO heart, which was associated with decreased cardiac contractility. The number of chromaffin cells in the adrenal medulla was also decreased, indicating a broad dependence on HIF-1α for development of the sympathetic nervous system.

Neuropilins guide preganglionic sympathetic axons and chromaffin cell precursors to establish the adrenal medulla.

The adrenal medulla is composed of neuroendocrine chromaffin cells that secrete adrenaline into the systemic circulation to maintain physiological homeostasis and enable the autonomic stress response. How chromaffin cell precursors colonise the adrenal medulla and how they become connected to central nervous system-derived preganglionic sympathetic neurons remain largely unknown. By combining lineage tracing, gene expression studies, genetic ablation and the analysis of mouse mutants, we demonstrate that preganglionic axons direct chromaffin cell precursors into the adrenal primordia. We further show that preganglionic axons and chromaffin cell precursors require class 3 semaphorin (SEMA3) signalling through neuropilins (NRP) to target the adrenal medulla. Thus, SEMA3 proteins serve as guidance cues to control formation of the adrenal neuroendocrine system by establishing appropriate connections between preganglionic neurons and adrenal chromaffin cells that regulate the autonomic stress response.

Spatial and activity-dependent catecholamine release in rat adrenal medulla under native neuronal stimulation.

Neuroendocrine chromaffin cells of the adrenal medulla in rat receive excitatory synaptic input through anterior and posterior divisions of the sympathetic splanchnic nerve. Upon synaptic stimulation, the adrenal medulla releases the catecholamines, epinephrine, and norepinephrine into the suprarenal vein for circulation throughout the body. Under sympathetic tone, catecholamine release is modest. However, upon activation of the sympathoadrenal stress reflex, and increased splanchnic firing, adrenal catecholamine output increases dramatically. Moreover, specific stressors can preferentially increase release of either epinephrine (i.e., hypoglycemia) or norepinephrine (i.e., cold stress). The mechanism for this stressor-dependent segregated release of catecholamine species is not yet fully understood. We tested the hypothesis that stimulation of either division of the splanchnic selects for epinephrine over norepinephrine release. We introduce an ex vivo rat preparation that maintains native splanchnic innervation of the adrenal gland and we document experimental advantages and limitations of this preparation. We utilize fast scanning cyclic voltammetry to detect release of both epinephrine and norepinephrine from the adrenal medulla, and report that epinephrine and norepinephrine release are regulated spatially and in a frequency-dependent manner. We provide data to show that epinephrine is secreted preferentially from the periphery of the medulla and exhibits a higher threshold and steeper stimulus-secretion function than norepinephrine. Elevated stimulation of the whole nerve specifically enhances epinephrine release from the peripheral medulla. Our data further show that elimination of either division from stimulation greatly attenuated epinephrine release under elevated stimulation, while either division alone can largely support norepinephrine release.

Hydralazine administration activates sympathetic preganglionic neurons whose activity mobilizes glucose and increases cardiovascular function.

Hypotensive drugs have been used to identify central neurons that mediate compensatory baroreceptor reflex responses. Such drugs also increase blood glucose. Our aim was to identify the neurochemical phenotypes of sympathetic preganglionic neurons (SPN) and adrenal chromaffin cells activated following hydralazine (HDZ; 10mg/kg) administration in rats, and utilize this and SPN target organ destination to ascribe their function as cardiovascular or glucose regulating. Blood glucose was measured and adrenal chromaffin cell activation was assessed using c-Fos immunoreactivity (-ir) and phosphorylation of tyrosine hydroxylase, respectively. The activation and neurochemical phenotype of SPN innervating the adrenal glands and celiac ganglia were determined using the retrograde tracer cholera toxin B subunit, in combination with in situ hybridization and immunohistochemistry. Blood glucose was elevated at multiple time points following HDZ administration but little evidence of chromaffin cell activation was seen suggesting non-adrenal mechanisms contribute to the sustained hyperglycemia. 16±0.1% of T4-T11 SPN contained c-Fos and of these: 24.3±1.4% projected to adrenal glands and 29±5.5% projected to celiac ganglia with the rest innervating other targets. 62.8±1.4% of SPN innervating adrenal glands were activated and 29.9±3.3% expressed PPE mRNA whereas 53.2±8.6% of SPN innervating celiac ganglia were activated and 31.2±8.8% expressed PPE mRNA. CART-ir SPN innervating each target were also activated and did not co-express PPE mRNA. Neurochemical coding reveals that HDZ administration activates both PPE+SPN, whose activity increase glucose mobilization causing hyperglycemia, as well as CART+SPN whose activity drive vasomotor responses mediated by baroreceptor unloading to raise vascular tone and heart rate.

Lineage and stage specific requirement for Dicer1 in sympathetic ganglia and adrenal medulla formation and maintenance.

The development of sympathetic neurons and chromaffin cells is differentially controlled at distinct stages by various extrinsic and intrinsic signals. Here we use conditional deletion of Dicer1 in neural crest cells and noradrenergic neuroblasts to identify stage specific functions in sympathoadrenal lineages. Conditional Dicer1 knockout in neural crest cells of Dicer1(Wnt1Cre) mice results in a rapid reduction in the size of developing sympathetic ganglia and adrenal medulla. In contrast, Dicer1 elimination in noradrenergic neuroblasts of Dicer1(DbhiCre) animals affects sympathetic neuron survival starting at late embryonic stages and chromaffin cells persist at least until postnatal week 1. A differential function of Dicer1 signaling for the development of embryonic noradrenergic and cholinergic sympathetic neurons is demonstrated by the selective increase in the expression of Tlx3 and the cholinergic marker genes VAChT and ChAT at E16.5. The number of Dbh, Th and TrkA expressing noradrenergic neurons is strongly decreased in Dicer1-deficient sympathetic ganglia at birth, whereas Tlx3(+)/ Ret(+) cholinergic neurons cells are spared from cell death. The postnatal death of chromaffin cells is preceded by the loss of Ascl1, mir-375 and Pnmt and an increase in the markers Ret and NF-M, which suggests that Dicer1 is required for the maintenance of chromaffin cell differentiation and survival. Taken together, these findings demonstrate distinct stage and lineage specific functions of Dicer1 signaling in differentiation and survival of sympathetic neurons and adrenal chromaffin cells.

Ontogeny of O2 and CO2//H+ chemosensitivity in adrenal chromaffin cells: role of innervation.

The adrenal medulla plays a key role in the physiological responses of developing and mature mammals by releasing catecholamines (CAT) during stress. In rodents and humans, the innervation of CAT-producing, adrenomedullary chromaffin cells (AMCs) is immature or absent during early postnatal life, when these cells possess 'direct' hypoxia- and CO2/H(+)-chemosensing mechanisms. During asphyxial stressors at birth, these mechanisms contribute to a CAT surge that is critical for adaptation to extra-uterine life. These direct chemosensing mechanisms regress postnatally, in parallel with maturation of splanchnic innervation. Here, we review the evidence that neurotransmitters released from the splanchnic nerve during innervation activate signaling cascades that ultimately cause regression of direct AMC chemosensitivity to hypoxia and hypercapnia. In particular, we consider the roles of cholinergic and opioid receptor signaling, given that splanchnic nerves release acetylcholine and opiate peptides onto their respective postsynaptic nicotinic and opioid receptors on AMCs. Recent in vivo and in vitro studies in the rat suggest that interactions involving α7 nicotinic acetylcholine receptors (nAChRs), the hypoxia inducible factor (HIF)-2α signaling pathway, protein kinases and ATP-sensitive K(+) (KATP) channels contribute to the selective suppression of hypoxic chemosensitivity. In contrast, interactions involving µ- and/or δ-opiod receptor signaling pathways contribute to the suppression of both hypoxic and hypercapnic chemosensitivity, via regulation of the expression of KATP channels and carbonic anhydrase (CA I and II), respectively. These data suggest that the ontogeny of O2 and CO2/H(+) chemosensitivity in chromaffin cells can be regulated by the tonic release of presynaptic neurotransmitters.

Brain RVD-haemopressin, a haemoglobin-derived peptide, inhibits bombesin-induced central activation of adrenomedullary outflow in the rat.

BACKGROUND AND PURPOSE: Haemopressin and RVD-haemopressin, derived from the haemoglobin α-chain, are bioactive peptides found in brain and are ligands for cannabinoid CB1 receptors. Activation of brain CB1 receptors inhibited the secretion of adrenal catecholamines (noradrenaline and adrenaline) induced by i.c.v. bombesin in the rat. Here, we investigated the effects of two haemoglobin-derived peptides on this bombesin-induced response EXPERIMENTAL APPROACH: Anaesthetised male Wistar rats were pretreated with either haemoglobin-derived peptide, given i.c.v., 30 min before i.c.v. bombesin and plasma catecholamines were subsequently measured electrochemically after HPLC. Direct effects of bombesin on secretion of adrenal catecholamines were examined using bovine adrenal chromaffin cells. Furthermore, activation of haemoglobin α-positive spinally projecting neurons in the rat hypothalamic paraventricular nucleus (PVN, a regulatory centre of central adrenomedullary outflow) after i.c.v. bombesin was assessed by immunohistochemical techniques. KEY RESULTS: Bombesin given i.c.v. dose-dependently elevated plasma catecholamines whereas incubation with bombesin had no effect on spontaneous and nicotine-induced secretion of catecholamines from chromaffin cells. The bombesin-induced increase in catecholamines was inhibited by pretreatment with i.c.v. RVD-haemopressin (CB1 receptor agonist) but not after pretreatment with haemopressin (CB1 receptor inverse agonist). Bombesin activated haemoglobin α-positive spinally projecting neurons in the PVN. CONCLUSIONS AND IMPLICATIONS: The haemoglobin-derived peptide RVD-haemopressin in the brain plays an inhibitory role in bombesin-induced activation of central adrenomedullary outflow via brain CB1 receptors in the rat. These findings provide basic information for the therapeutic use of haemoglobin-derived peptides in the modulation of central adrenomedullary outflow.

Spinal cord injury level and the circulating cytokine response to strenuous exercise.

PURPOSE: A complete spinal cord injury (SCI) above the sixth thoracic vertebra (T6) results in the loss of sympathetic innervation of the adrenal medulla. This study examined the effect of a complete SCI above and below T6 on plasma concentrations of epinephrine, circulating interleukin 6 (IL-6) and other inflammatory cytokines in response to acute strenuous exercise. METHODS: Twenty-six elite male wheelchair athletes (8 = C6-C7 tetraplegic [TETRA], 10 = T6-L1 paraplegic [PARA], and 8 = non-spinal-cord-injured controls [NON-SCI]) performed a submaximal exercise test followed by a graded exercise to exhaustion on a motorized treadmill. Blood samples were taken preexercise, postexercise, and 30 min postexercise and analyzed for concentrations of IL-6, IL-10, IL-1 receptor antagonist (IL-1ra), tumor necrosis factor α (TNF-α), epinephrine, and cortisol. RESULTS: The circulating IL-6 concentration was significantly elevated at postexercise and 30 min postexercise (post30; approximately fivefold) in NON-SCI and PARA (P = 0.003), whereas concentrations in TETRA did not change significantly from preexercise values. IL-10, IL-1ra, and TNF-α were unaffected by exercise in all groups; however, both SCI groups presented elevated concentrations of IL-10 compared with NON-SCI (P = 0.001). At postexercise, epinephrine concentrations were significantly higher than preexercise and post30 concentrations in NON-SCI (approximately threefold) and PARA (approximately twofold) (P = 0.02). Plasma epinephrine concentrations were unchanged in TETRA throughout exercise; concentrations were significantly lower than NON-SCI and PARA at all time points. Plasma cortisol concentrations were significantly elevated in all groups at postexercise and post30 compared with preexercise (P < 0.001). Total exercise time was similar between groups (NON-SCI = 38 ± 6; PARA = 35 ± 5; TETRA = 36 ± 5 min). CONCLUSIONS: These findings suggest that the sympathetic nervous system plays an important regulatory role in the circulating IL-6 response to exercise and has implications for the metabolic and inflammatory responses to exercise in individuals with injuries above T6.

Ca2+ imaging in perfused adrenal medullae.

The Ca2+ imaging method was developed to explore changes in excitability in adrenal medullary (AM) cells in a large field in response to synaptic input and chemicals. The adrenal medullae of rats and guinea pigs were retrogradely loaded with Ca2+ indicator through the adrenal vein. Nerve fibers remaining in the adrenal gland were electrically stimulated to induce postsynaptic responses in AM cells, and chemicals were applied to the cells by adding to the perfusate. With this method, gamma-aminobutyric acid (GABA) was shown to increase the Ca2+ signal in almost all and 40% AM cells in guinea pigs and rats, respectively.

Sympathetic innervation of the splanchnic region mediates the beneficial hemodynamic effects of 8-OH-DPAT in hemorrhagic shock.

Administration of the 5-HT(1A) receptor agonist, 8-OH-DPAT, improves cardiovascular hemodynamics and tissue oxygenation in conscious rats subjected to hypovolemic shock. This effect is mediated by sympathetic-dependent increases in venous tone. To determine the role of splanchnic nerves in this response, effects of 8-OH-DPAT (30 nmol/kg iv) were measured following fixed-arterial blood pressure hemorrhagic shock (i.e., maintenance of 50 mmHg arterial pressure for 25 min) in rats subjected to bilateral splanchnic nerve denervation (SD). Splanchnic denervation decreased baseline venous tone as measured by mean circulatory filling pressure (MCFP) and accelerated the onset of hypotension during blood loss. Splanchnic denervation did not affect the immediate pressor effect of 8-OH-DPAT but did reverse the drug's lasting pressor effect, as well as its ability to increase MCFP and improve metabolic acidosis. Like SD, adrenal demedullation (ADMX) lowered baseline MCFP and accelerated the hypotensive response to blood withdrawal but also reduced the volume of blood withdrawal required to maintain arterial blood pressure at 50 mmHg. 8-OH-DPAT raised MCFP early after administration in ADMX rats, but the response did not persist throughout the posthemorrhage period. In a fixed-volume hemorrhage model, 8-OH-DPAT continued to raise blood pressure in ADMX rats. However, it produced only a transient and variable rise in MCFP compared with sham-operated animals. The data indicate that 8-OH-DPAT increases venoconstriction and improves acid-base balance in hypovolemic rats through activation of splanchnic nerves. This effect is due, in part, to activation of the adrenal medulla.

Differential control of efferent sympathetic activity revisited.

This article reviews 40 years of research (1970-2010) into the capability of the efferent sympathetic nervous system to display differential responsiveness. Discovered first were antagonistic changes of activity in sympathetic filaments innervating functionally different sections of the cardiovascular system in response to thermal stimulation. During the subsequent four decades of investigation, a multitude of differential sympathetic efferent response patterns were identified, ranging from opposing activity changes at the level of multi-fiber filaments innervating different organs to the level of single fibers controlling functionally different structures in the same organ. Differential sympathetic responsiveness was shown to be displayed in response to exogenous or artificial stimulation of afferent sensory fibers transmitting particular exogenous stimuli, especially those activating peripheral nociceptors. Moreover, sympathetic differentiation was found to be characteristic of autonomic responses to environmental changes by which homeostasis in the broadest sense would be challenged. Heat or cold loads or their experimental equivalents, altered composition of inspired air or changes in blood gas composition, imbalances of body fluid control, and exposure to agents challenging the immune system were shown to elicit differential efferent sympathetic response patterns which often displayed a high degree of specificity. In summary, autonomic adjustments to changes of biometeorological parameters may be considered as representative of the capability of the sympathetic nervous system to exert highly specific efferent control of organ functions by which bodily homeostasis is maintained.

[The immunomorphologic analysis of innervation of chromaffin paraganglian cells of the mammalian arteries and heart].

Innervation of chromaffin cells of paraganglia of the wall of mammalian large arterial vessels and heart (in rat, cat, and human) was studied by neuromorphological and immunohistochemical methods. There is established similarity in structure of specialized, "basket"-type nerve endings of the chromaffin cells (ChC) with pericellular nerve apparatuses of sympathetic and parasympathetic autonomic neurons. It is proposed to use immunohistochemical reaction for synaptophysin as method of selective detection of ChC of paraganglia and adrenal medulla. The conclusion is made that synaptophysin-positive terminals (SPPT) found on bodies of ChC and postganglionic neurons represent efferent, rather than afferent, synapses formed by myelinated axons of preganglionic fibers. It is suggested that ChC of paraganglia alongside with their characteristic endocrine function participate in complex mechanisms of chemoreceptor regulation of tissue homeostasis of mammalian blood vessels and heart.

Neural and humoral control of regional vascular beds via A1 adenosine receptors located in the nucleus tractus solitarii.

Our previous studies showed that stimulation of adenosine A(1) receptors located in the nucleus of the solitary tract (NTS) exerts counteracting effects on the iliac vascular bed: activation of the adrenal medulla and ß-adrenergic vasodilation vs. sympathetic and vasopressinergic vasoconstriction. Because NTS A(1) adenosine receptors inhibit baroreflex transmission in the NTS and contribute to the pressor component of the HDR, we hypothesized that these receptors also contribute to the redistribution of blood from the visceral to the muscle vasculature via prevailing sympathetic and vasopressinergic vasoconstriction in the visceral (renal and mesenteric) vascular beds and prevailing ß-adrenergic vasodilation in the somatic (iliac) vasculature. To test this hypothesis, we compared the A(1) adenosine-receptor-mediated effects of each vasoactive factor triggered by NTS A(1) adenosine receptor stimulation [N(6)-cyclopentyladenosine (CPA), 330 pmol in 50 nl] on the regional vascular responses in urethane/chloralose-anesthetized rats. The single-factor effects were separated using adrenalectomy, ß-adrenergic blockade, V(1) vasopressin receptor blockade, and sinoaortic denervation. In intact animals, initial vasodilation was followed by large, sustained vasoconstriction with smaller responses observed in renal vs. mesenteric and iliac vascular beds. The initial ß-adrenergic vasodilation prevailed in the iliac vs. mesenteric and renal vasculature. The large and sustained vasopressinergic vasoconstriction was similar in all vascular beds. Small sympathetic vasoconstriction was observed only in the iliac vasculature in this setting. We conclude that, although A(1) adenosine-receptor-mediated ß-adrenergic vasodilation may contribute to the redistribution of blood from the visceral to the muscle vasculature, this effect is overridden by sympathetic and vasopressinergic vasoconstriction.

Neuropeptide coding of sympathetic preganglionic neurons; focus on adrenally projecting populations.

Chemical coding of sympathetic preganglionic neurons (SPN) suggests that the chemical content of subpopulations of SPN can define their function. Since neuropeptides, once synthesized are transported to the axon terminal, most demonstrated chemical coding has been identified using immunoreactive terminals at the target organ. Here, we use a different approach to identify and quantify the subpopulations of SPN that contain the mRNA for pituitary adenylate cyclase activating polypeptide (PACAP) or enkephalin. Using double-labeled immunohistochemistry combined with in situ hybridization (ISH) we firstly identified the distribution of these mRNAs in the spinal cord and determined quantitatively, in Sprague-Dawley rats, that many SPN at the T4-T10 spinal level contain preproPACAP (PPP+, 80 ± 3%, n=3), whereas a very small percentage contain preproenkephalin (PPE+, 4 ± 2%, n=4). A similar neurochemical distribution was found at C8-T3 spinal level. These data suggest that PACAP potentially regulates a large number of functions dictated by SPN whereas enkephalins are involved in few functions. We extended the study to explore those SPN that control adrenal chromaffin cells. We found 97 ± 5% of adrenally projecting SPN (AP-SPN) to be PPP+ (n=4) with only 47 ± 3% that were PPE+ (n=5). These data indicate that adrenally projecting PACAPergic SPN regulate both adrenal adrenaline (Ad) and noradrenaline (NAd) release whereas the enkephalinergic SPN subpopulation must control a (sub) population of chromaffin cells - most likely those that release Ad. The sensory innervation of the adrenal gland was also determined. Of the few adrenally projecting dorsal root ganglia (AP-DRG) observed, 74 ± 12% were PPP+ (n=3), whereas 1 ± 1% were PPE+ (n=3). Therefore, if sensory neurons release peptides to the adrenal medulla, PACAP is most likely involved. Together, these data provide a neurochemical basis for differential control of sympathetic outflow particularly that to the adrenal medulla.

Dual effects of nobiletin, a citrus polymethoxy flavone, on catecholamine secretion in cultured bovine adrenal medullary cells.

Nobiletin, a compound of polymethoxy flavones found in citrus fruits, possesses a wide range of pharmacological activities. Here we report the effects of nobiletin on catecholamine secretion in cultured bovine adrenal medullary cells. Nobiletin (1.0-100 microM) concentration-dependently stimulated catecholamine secretion and (45)Ca(2+) influx. Its stimulatory effect of nobiletin on catecholamine secretion was abolished by deprivation of extracellular Ca(2+) and partially inhibited by specific inhibitors of voltage-dependent Ca(2+) channels and Na(+)/Ca(2+) exchangers. On the other hand, nobiletin suppressed catecholamine secretion and (22)Na(+) and (45)Ca(2+) influx induced by acetylcholine, an agonist of nicotinic acetylcholine receptors, in a concentration-dependent manner. It also inhibited catecholamine secretion, (22)Na(+) influx and/or (45)Ca(2+) influx induced by veratridine, an activator of voltage-dependent Na(+) channels, and 56 mM K(+), an activator of voltage-dependent Ca(2+) channels. In Xenopus oocytes expressing alpha3beta4 neuronal acetylcholine receptors, nobiletin directly inhibited the current evoked by acetylcholine in a concentration-dependent manner similar to that observed in catecholamine secretion. The present findings suggest that nobiletin, by itself, stimulates catecholamine secretion via activation of voltage-dependent Ca(2+) channels or Na(+)/Ca(2+) exchangers, whereas it inhibits catecholamine secretion induced by acetylcholine through the suppression of Na(+) influx and Ca(2+) influx in cultured bovine adrenal medullary cells.

Obesity and catecholamine responses to maximal exercise in adolescent girls.

The aim of this study was to investigate plasma catecholamine [adrenaline (A) and noradrenaline (NA)] concentrations at rest and in response to maximal exercise in three different groups of adolescent girls. According to their body mass index, 34 adolescent girls aged 15-16 years were divided into three groups: a normal weight group (NO) (n = 11), an overweight group (OW) (n = 11) and an obese group (OB) (n = 12). Plasma A and NA concentrations were measured at rest during fasting conditions (A(0) and NA(0)), after a standardized breakfast (A(rest) and NA(rest)) and immediately after an incremental exhaustive exercise (A(EX) and NA(EX)). A (0) and NA(0) were not significantly different among the three groups. However, the A(0)/NA(0) was statistically lower in OB compared to OW and NO. A(EX) and NA(EX) were significantly higher than resting values in the three groups. However, in response to exercise, no significant differences were reported between OB (A(EX) = 2.20 +/- 0.13 nmol/l, NA(EX) = 12.28 +/- 0.64 nmol/l), OW (A(EX) = 2.39 +/- 0.23 nmol/l, NA(EX) = 12.94 +/- 0.93 nmol/l) and NO (A(EX) = 2.52 +/- 0.24 nmol/l, NA(EX) = 12.60 +/- 0.63 nmol/l). In conclusion, our results showed that at rest, in adolescent girls, the responsiveness of the adrenal medulla to the sympathetic nervous activity is lower in OB subjects compared to OW and NO ones. However, in response to maximal exercise, plasma catecholamines are not affected by obesity.

Electrophysiological and morphological features underlying neurotransmission efficacy at the splanchnic nerve-chromaffin cell synapse of bovine adrenal medulla.

The ability of adrenal chromaffin cells to fast-release catecholamines relies on their capacity to fire action potentials (APs). However, little attention has been paid to the requirements needed to evoke the controlled firing of APs. Few data are available in rodents and none on the bovine chromaffin cell, a model extensively used by researchers. The aim of this work was to clarify this issue. Short puffs of acetylcholine (ACh) were fast perifused to current-clamped chromaffin cells and produced the firing of single APs. Based on the currents generated by such ACh applications and previous literature, current waveforms that efficiently elicited APs at frequencies up to 20 Hz were generated. Complex waveforms were also generated by adding simple waveforms with different delays; these waveforms aimed at modeling the stimulation patterns that a chromaffin cell would conceivably undergo upon strong synaptic stimulation. Cholinergic innervation was assessed using the acetylcholinesterase staining technique on the supposition that the innervation pattern is a determinant of the kind of stimuli chromaffin cells can receive. It is concluded that 1) a reliable method to produce frequency-controlled APs by applying defined current injection waveforms is achieved; 2) the APs thus generated have essentially the same features as those spontaneously emitted by the cell and those elicited by fast-ACh perifusion; 3) the higher frequencies attainable peak at around 30 Hz; and 4) the bovine adrenal medulla shows abundant cholinergic innervation, and chromaffin cells show strong acetylcholinesterase staining, consistent with a tight cholinergic presynaptic control of firing frequency.

Morphological and microvascular changes of the adrenal glands in streptozotocin-induced long-term diabetic rats.

It has been known that diabetes mellitus is associated with hyperfunction of the adrenal gland. However, the structural changes of adrenal gland in diabetes have rarely been studied. The aims of this study were to investigate the morphological and microvascular alterations in streptozotocin (STZ)-induced long-term diabetic rats. Twelve male Sprague-Dawley rats were divided into diabetic (n=8) and control (n=4) groups. Each diabetic rat was induced by an intraperitoneal injection of STZ (60 mg/kg) in citrate buffer (pH 4.5). Control rats were intraperitoneally injected with the same amounts of the buffer. These animals were sacrificed at 20 weeks after the injections. The adrenal glands were processed for the morphological and microvascular studies by using conventional light microscopy (LM) and vascular corrosion cast technique combined with scanning electron microscopy (SEM), respectively. In the diabetic group, the cells in zona glomeruloza (ZG) became atrophied and the thickness of this zone was found to be less than that of the controls. In the zona fasciculata (ZF) and zona reticularis (ZR), the hypertrophic cells were investigated in both layers. The degenerated chromaffin and hypertrophic sympathetic ganglion cells in the adrenal medulla were observed. Also some degenerated ganglion cells were found. Additionally, lymphocyte infiltration, macrophages and amyloidosis were found in the adrenal medulla of long-term diabetic rats with renal failure. Under the SEM observation, the luminal diameters of capillaries in the diabetic group were dilated in all zones. In addition, these capillaries in the ZF and ZR were arranged in tortuous courses. This study demonstrates morphological and microvascular changes in the adrenal gland of diabetic rats which are in accordance with the hormonal changes reported by previous investigators.