Adrenal chromaffin cells (ACCs) are the neuroendocrine arm of the sympathetic nervous system and key mediators of the physiological stress response. Acetylcholine (ACh) released from preganglionic splanchnic nerves activates nicotinic acetylcholine receptors (nAChRs) on chromaffin cells causing membrane depolarization, opening voltage-gated Ca2+ channels (VGCC), and exocytosis of catecholamines and neuropeptides. The serotonin transporter is expressed in ACCs and interacts with 5-HT1A receptors to control secretion. In addition to blocking the serotonin transporter, some selective serotonin reuptake inhibitors (SSRIs) are also agonists at sigma-1 receptors which function as intracellular chaperone proteins and can translocate to the plasma membrane to modulate ion channels. Therefore, we investigated whether SSRIs and other sigma-1 receptor ligands can modulate stimulus-secretion coupling in ACCs. Escitalopram and fluvoxamine (100 nM to 1 μM) reversibly inhibited nAChR currents. The sigma-1 receptor antagonists NE-100 and BD-1047 also blocked nAChR currents (≈ 50% block at 100 nM) as did PRE-084, a sigma-1 receptor agonist. Block of nAChR currents by fluvoxamine and NE-100 was not additive suggesting a common site of action. VGCC currents were unaffected by the drugs. Neither the increase in cytosolic [Ca2+ ] nor the resulting catecholamine secretion evoked by direct membrane depolarization to bypass nAChRs was altered by fluvoxamine or NE-100. However, both Ca2+ entry and catecholamine secretion evoked by the cholinergic agonist carbachol were significantly reduced by fluvoxamine or NE-100. Together, our data suggest that sigma-1 receptors do not acutely regulate catecholamine secretion. Rather, SSRIs and other sigma-1 receptor ligands inhibit secretion evoked by cholinergic stimulation because of direct block of Ca2+ entry via nAChRs.

Stimulation of the lateral hypothalamus (LH) produces antinociception that is modified by intrathecal alpha-adrenergic antagonists. Spinally-projecting noradrenergic neurons in the LH have not been identified, suggesting that the LH may innervate brainstem noradrenergic neurons, such as the A7 catecholamine cell group in the dorsolateral pontine tegmentum, that modify nociception at the level of the spinal cord dorsal horn. Recently we demonstrated in neuroanatomical studies that substance P-immunoreactive neurons in the LH project the A7 area. To identify a functional connection between substance P neurons in the LH and the A7 cell group, the cholinergic agonist carbachol (125 nmol) was microinjected into the LH of female Sprague-Dawley rats and antinociception was obtained on the tail flick or foot withdrawal test. Cobalt chloride (100 nM) was then microinjected near the A7 cell group to block synaptic activation of spinally-projecting A7 neurons, which were identified using tyrosine-hydroxylase immunoreactivity. Within 5 min of the cobalt chloride injection, the antinociceptive effect of carbachol stimulation was blocked. In another set of experiments, the NK(1) receptor antagonist L-703-606 (5 microg) was microinjected near the A7 cell group following LH stimulation with carbachol. L-703-606 also abolished LH-induced antinociception. These results support the conclusion that antinociception produced by activating substance P neurons in the LH is mediated in part by the subsequent activation of spinally-projecting noradrenergic neurons in the A7 cell group.

Bivalve metamorphosis is a developmental transition from a free-living larva to a benthic juvenile (spat), regulated by a complex interaction of neurotransmitters and neurohormones such as L-DOPA and epinephrine (catecholamine). We recently suggested an N-Methyl-D-aspartate (NMDA) receptor pathway as an additional and previously unknown regulator of bivalve metamorphosis. To explore this theory further, we successfully induced metamorphosis in the Pacific oyster, Crassostrea gigas, by exposing competent larvae to L-DOPA, epinephrine, MK-801 and ifenprodil. Subsequently, we cloned three NMDA receptor subunits CgNR1, CgNR2A and CgNR2B, with sequence analysis suggesting successful assembly of functional NMDA receptor complexes and binding to natural occurring agonists and the channel blocker MK-801. NMDA receptor subunits are expressed in competent larvae, during metamorphosis and in spat, but this expression is neither self-regulated nor regulated by catecholamines. In-situ hybridisation of CgNR1 in competent larvae identified NMDA receptor presence in the apical organ/cerebral ganglia area with a potential sensory function, and in the nervous network of the foot indicating an additional putative muscle regulatory function. Furthermore, phylogenetic analyses identified molluscan-specific gene expansions of key enzymes involved in catecholamine biosynthesis. However, exposure to MK-801 did not alter the expression of selected key enzymes, suggesting that NMDA receptors do not regulate the biosynthesis of catecholamines via gene expression.

Function of nicotine, which induces activation of all parts of the body including our brain, has been receiving much attention for a long period of time and also been actively studied by researchers for its pharmacological actions in the central nervous system. The modulation of nicotine concentration and the inhibition of nicotine binding on target receptors in the brain are the key factors for smoking addiction therapy. In previous studies showed that influx of nicotine at the blood-brain barrier was through the pyrilamine-sensitive organic cation transporters. But the direct interacting mechanism of pyrilamine on the nicotine binding target receptors has not yet been clarified. The aim of the present study is to investigate the direct binding mechanisms of a pyrilamine on the nicotinic acetylcholine receptors (nAChRs). We found that pyrilamine shares the same ligand binding pocket of nicotine (NCT) on nAChRs but interacts with more amino acid residues than NCT does. The extended part of pyrilamine interacts with additional residues in the ligand binding pocket of nAChRs which are located nearby the entrance of the binding pocket. The catecholamine (CA) secretion induced by nAChR agonist (NCT') was significantly inhibited by the pyrilamine pretreatment. Real time carbon-fiber amperometry confirmed the inhibition of the NCT'-induced exocytosis by pyrilamine in a single cell level. We also found that pyrilamine inhibited the NCT'-induced [Ca(2+)]i. In contrast, pyrilamine did not affect the increase in calcium induced by high K(+). Overall, these data suggest that pyrilamine directly docks into the ligand binding site of nAChRs and specifically inhibits the nAChR-mediated effects thereby causing inhibition of CA secretion. Therefore, pyrilamine may play an important role to explore new treatments to aid smoking cessation.

The present study was designed to investigate the characteristics of gintonin, one of components isolated from Korean Ginseng on secretion of catecholamines (CA) from the isolated perfused model of rat adrenal gland and to clarify its mechanism of action. Gintonin (1 to 30 µg/ml), perfused into an adrenal vein, markedly increased the CA secretion from the perfused rat adrenal medulla in a dose-dependent fashion. The gintonin-evoked CA secretion was greatly inhibited in the presence of chlorisondamine (1 µM, an autonomic ganglionic bloker), pirenzepine (2 µM, a muscarinic M1 receptor antagonist), Ki14625 (10 µM, an LPA1/3 receptor antagonist), amiloride (1 mM, an inhibitor of Na+/Ca2+ exchanger), a nicardipine (1 µM, a voltage-dependent Ca2+ channel blocker), TMB-8 (1 µM, an intracellular Ca2+ antagonist), and perfusion of Ca2+-free Krebs solution with 5mM EGTA (a Ca2+chelater), while was not affected by sodium nitroprusside (100 µM, a nitrosovasodialtor). Interestingly, LPA (0.3~3 µM, an LPA receptor agonist) also dose-dependently enhanced the CA secretion from the adrenal medulla, but this facilitatory effect of LPA was greatly inhibited in the presence of Ki 14625 (10 µM). Moreover, acetylcholine (AC)-evoked CA secretion was greatly potentiated during the perfusion of gintonin (3 µg/ml). Taken together, these results demonstrate the first evidence that gintonin increases the CA secretion from the perfused rat adrenal medulla in a dose-dependent fashion. This facilitatory effect of gintonin seems to be associated with activation of LPA- and cholinergic-receptors, which are relevant to the cytoplasmic Ca2+ increase by stimulation of the Ca2+ influx as well as by the inhibition of Ca2+ uptake into the cytoplasmic Ca2+ stores, without the increased nitric oxide (NO). Based on these results, it is thought that gintonin, one of ginseng components, can elevate the CA secretion from adrenal medulla by regulating the Ca2+ mobilization for exocytosis, suggesting facilitation of cardiovascular system. Also, these findings show that gintonin might be at least one of ginseng-induced hypertensive components.

Melatonin is functionally involved in the control of circadian rhythm and hormonal secretion. In the present study, we investigated the roles of melatonin in the interaction of catecholamine synthesis with adrenocortical steroids by focusing on bone morphogenetic protein (BMP)-4 expressed in the adrenal medulla using rat pheochromocytoma PC12 cells. Melatonin treatment significantly reduced the mRNA expression of catecholamine synthases, including the rate-limiting enzyme tyrosine hydroxylase (Th), 3,4-dihydroxyphenylalanine decarboxylase and dopamine-β-hydroxylase expressed in PC12 cells. In accordance with changes in the expression levels of enzymes, dopamine production and cAMP synthesis determined in the culture medium and cell lysate were also suppressed by melatonin. The MT1 receptor, but not the MT2 receptor, was expressed in PC12 cells, and luzindole treatment reversed the inhibitory effect of melatonin on Th expression, suggesting that MT1 is a functional receptor for the control of catecholamine synthesis. Interestingly, melatonin enhanced the inhibitory effect of BMP-4 on Th mRNA expression in PC12 cells. Melatonin treatment accelerated BMP-4-induced phosphorylation of SMAD1/5/8 and transcription of the BMP target gene Id1. Of note, melatonin significantly upregulated Alk2 and Bmpr2 mRNA levels but suppressed inhibitory Smad6/7 expression, leading to the enhancement of SMAD1/5/8 signaling in PC12 cells, while BMP-4 did not affect Mt1 expression. Regarding the interaction with adrenocortical steroids, melatonin preferentially enhanced glucocorticoid-induced Th mRNA through upregulation of the glucocorticoid receptor and downregulation of Bmp4 expression, whereas melatonin repressed Th mRNA expression induced by aldosterone or androgen without affecting expression levels of the receptors for mineralocorticoid and androgen. Collectively, the results indicate that melatonin plays a modulatory role in catecholamine synthesis by cooperating with BMP-4 and glucocorticoid in the adrenal medulla.

In an effort to determine mechanisms of action of the putative anti-addictive agent ibogaine, we have measured its effects on catecholamine release in a model neuronal system, cultured bovine chromaffin cells. Various modes of stimulating catecholamine release were used including nicotinic ACh receptor activation, membrane depolarization with elevated K+ and Na+ channel activation with veratridine. In addition, because ibogaine has been reported to interact with kappa opioid receptors, we tested whether kappa receptor antagonists could reverse ibogaine's effects on catecholamine release. Ibogaine, at low concentration (<10 microM) was found to selectively inhibit nicotinic receptor-mediated catecholamine release, while having no significant effect on release evoked by either veratridine or membrane depolarization with elevated K+. The inhibitory actions of ibogaine and the kappa agonists were not reversed by preincubation with the opioid antagonists nor-binaltorphimine or naltrexone, suggesting that these inhibitory effects are not mediated by the kappa opioid receptor. The effects of low dose (10 microM) ibogaine were rapidly reversible, while the inhibitory effects of higher ibogaine doses persisted for at least 19 h following ibogaine washout. The results provide evidence for a mechanism of action ibogaine at the nicotinic ACh receptor. The results are consistent with a model in which the initial high transient brain concentrations (100 microM) of ibogaine act at multiple cellular sites and then have a selective action at the nicotinic ACh receptor cation channel following its metabolism to lower brain concentrations. The present findings are relevant to potential anti-addictive actions of ibogaine and to the development of drugs to combat nicotine addiction.

Stimulation of adipocyte β-adrenergic receptors (β-ARs) induces expression of uncoupling protein 1 (UCP1), promoting nonshivering thermogenesis. Association of β-ARs with a lysine-myristoylated form of A kinase-anchoring protein 12 (AKAP12, also known as gravin-α) is required for downstream signaling that culminates in UCP1 induction. Conversely, demyristoylation of gravin-α by histone deacetylase 11 (HDAC11) suppresses this pathway. Whether inhibition of HDAC11 in adipocytes is sufficient to drive UCP1 expression independently of β-ARs is not known. Here, we demonstrate that adipocyte-specific deletion of HDAC11 in mice leads to robust induction of UCP1 in adipose tissue (AT), resulting in increased body temperature. These effects are mimicked by treating mice in vivo or human AT ex vivo with an HDAC11-selective inhibitor, FT895. FT895 triggers biphasic, gravin-α myristoylation-dependent induction of UCP1 protein expression, with a noncanonical acute response that is posttranscriptional and independent of protein kinase A (PKA), and a delayed response requiring PKA activity and new Ucp1 mRNA synthesis. Remarkably, HDAC11 inhibition promotes UCP1 expression even in models of adipocyte catecholamine resistance where β-AR signaling is blocked. These findings define cell-autonomous, multimodal roles for HDAC11 as a suppressor of thermogenesis, and highlight the potential of inhibiting HDAC11 to therapeutically alter AT phenotype independently of β-AR stimulation.

We examined if life-long mild caloric restriction (CR) alone or with voluntary exercise prevents the age-related changes in catecholamine biosynthetic enzyme levels in the adrenal medulla and hypothalamus. Ten-week-old Fisher-344 rats were assigned to: sedentary; sedentary+8% CR; or 8% CR+wheel running. Rats were euthanized at 6 or 24 months of age. Tyrosine hydroxylase (TH) mRNA expression was 4.4-fold higher in the adrenal medullae and 60% lower in the hypothalamus of old sedentary rats compared to young (p<0.01). Life-long CR reduced the age-related increase in adrenomedullary TH by 50% (p<0.05), and completely reversed the changes in hypothalamic TH. Voluntary exercise, however, had no additional effect over CR. Since angiotensin II is involved in the regulation of catecholamine biosynthesis, we examined the expressions of angiotensin II receptor subtypes in the adrenal medulla. AT(1) protein levels were 2.8-fold higher in the old animals compared to young (p<0.01), and while AT(1) levels were unaffected by CR alone, CR+wheel running decreased AT(1) levels by 50% (p<0.01). AT(2) levels did not change with age, however CR+wheel running increased its level by 42% (p<0.05). These data indicate that a small decrease in daily food intake can avert age-related changes in catecholamine biosynthetic enzyme levels in the adrenal medulla and hypothalamus, possibly through affecting angiotensin II signaling.

Stimulation of adipocyte β-adrenergic receptors (β-ARs) induces expression of uncoupling protein 1 (UCP1), promoting non-shivering thermogenesis. Association of β-ARs with a lysine myristoylated form of A-kinase anchoring protein 12 (AKAP12)/gravin-α is required for downstream signaling that culminates in UCP1 induction. Conversely, demyristoylation of gravin-α by histone deacetylase 11 (HDAC11) suppresses this pathway. Whether inhibition of HDAC11 in adipocytes is sufficient to drive UCP1 expression independently of β-ARs is not known. Here, we demonstrate that adipocyte-specific deletion of HDAC11 in mice leads to robust induction of UCP1 in adipose tissue (AT), resulting in increased body temperature. These effects are mimicked by treating mice in vivo or human AT ex vivo with an HDAC11-selective inhibitor, FT895. FT895 triggers biphasic, gravin-α myristoylation-dependent induction of UCP1 protein expression, with a non-canonical acute response that is post-transcriptional and independent of protein kinase A (PKA), and a delayed response requiring PKA activity and new Ucp1 mRNA synthesis. Remarkably, HDAC11 inhibition promotes UCP1 expression even in models of adipocyte catecholamine resistance where β-AR signaling is blocked. These findings define cell autonomous, multi-modal roles for HDAC11 as a suppressor of thermogenesis, and highlight the potential of inhibiting HDAC11 to therapeutically alter AT phenotype independently of β-AR stimulation.

Hindbrain catecholamine neurons are required for elicitation of feeding responses to glucose deficit, but the forebrain circuitry required for these responses is incompletely understood. Here we examined interactions of catecholamine and orexin neurons in eliciting glucoprivic feeding. Orexin neurons, located in the perifornical lateral hypothalamus (PeFLH), are heavily innervated by hindbrain catecholamine neurons, stimulate food intake, and increase arousal and behavioral activation. Orexin neurons may therefore contribute importantly to appetitive responses, such as food seeking, during glucoprivation. Retrograde tracing results showed that nearly all innervation of the PeFLH from the hindbrain originated from catecholamine neurons and some raphe nuclei. Results also suggested that many catecholamine neurons project collaterally to the PeFLH and paraventricular hypothalamic nucleus. Systemic administration of the antiglycolytic agent, 2-deoxy-D-glucose, increased food intake and c-Fos expression in orexin neurons. Both responses were eliminated by a lesion of catecholamine neurons innervating orexin neurons using the retrogradely transported immunotoxin, anti-dopamine-β-hydroxylase saporin, which is specifically internalized by dopamine-β-hydroxylase-expressing catecholamine neurons. Using designer receptors exclusively activated by designer drugs in transgenic rats expressing Cre recombinase under the control of tyrosine hydroxylase promoter, catecholamine neurons in cell groups A1 and C1 of the ventrolateral medulla were activated selectively by peripheral injection of clozapine-N-oxide. Clozapine-N-oxide injection increased food intake and c-Fos expression in PeFLH orexin neurons as well as in paraventricular hypothalamic nucleus neurons. In summary, catecholamine neurons are required for the activation of orexin neurons during glucoprivation. Activation of orexin neurons may contribute to appetitive responses required for glucoprivic feeding.

Disruption to endothelial cell homeostasis results in an extensive variety of human pathologies that are particularly relevant to major trauma. Circulating catecholamines, such as adrenaline and noradrenaline, activate endothelial adrenergic receptors triggering a potent response in endothelial function. The regulation of the endothelial cell metabolism is distinct and profoundly important to endothelium homeostasis. However, a precise catalogue of the metabolic alterations caused by sustained high catecholamine levels that results in endothelial dysfunction is still underexplored. Here, we uncover a set of up to 46 metabolites that exhibit a dose-response relationship to adrenaline-noradrenaline equimolar treatment. The identified metabolites align with the glutathione-ascorbate cycle and the nitric oxide biosynthesis pathway. Certain key metabolites, such as arginine and reduced glutathione, displayed a differential response to treatment in early (4 h) compared to late (24 h) stages of sustained stimulation, indicative of homeostatic metabolic feedback loops. Furthermore, we quantified an increase in the glucose consumption and aerobic respiration in endothelial cells upon catecholamine stimulation. Our results indicate that oxidative stress and nitric oxide metabolic pathways are downstream consequences of endothelial cell stimulation with sustained high levels of catecholamines. A precise understanding of the metabolic response in endothelial cells to pathological levels of catecholamines will facilitate the identification of more efficient clinical interventions in trauma patients.

Glucocorticoids are produced by the adrenal cortex and regulate cell metabolism in a variety of organs. This occurs either directly, by acting on specific receptors in a variety of cells, or by stimulating catecholamine expression within neighbor cells of the adrenal medulla. In this way, the whole adrenal gland may support specific metabolic requirements to cope with stressful conditions from external environment or internal organs. In addition, glucocorticoid levels may increase significantly in the presence of inappropriate secretion from adrenal cortex or may be administered at high doses to treat inflammatory disorders. In these conditions, metabolic alterations and increased blood pressure may occur, although altered sleep-waking cycle, anxiety, and mood disorders are frequent. These latter symptoms remain unexplained at the molecular level, although they overlap remarkably with disorders affecting catecholamine nuclei of the brainstem reticular formation. In fact, the present study indicates that various doses of glucocorticoids alter the expression of genes and proteins, which are specific for reticular catecholamine neurons. In detail, corticosterone administration to organotypic mouse brainstem cultures significantly increases Tyrosine hydroxylase (TH) and Dopamine transporter (DAT), while Phenylethanolamine N-methyltransferase (PNMT) is not affected. On the other hand, Dopamine Beta-Hydroxylase (DBH) increases only after very high doses of corticosterone.

For over 50 years, the sympathomimetic phenylpropanolamine (PPA; ±-norephedrine) was a primary active ingredient in over-the-counter nasal decongestants for both children and adults and continues to be prevalent in the vast majority of countries today. Previously, we reported that juvenile PPA exposure alters the developmental trajectory of catecholamine and amino acid neurotransmitter systems in the nucleus accumbens (NAC), impacting the motivational valence of cocaine in later life. The present study employed a combination of in vivo microdialysis and immunoblotting approaches to better understand how juvenile PPA exposure impacts catecholamine and glutamate function within the NAC. For this, C57BL/6J mice were pretreated repeatedly with PPA (0 or 40 mg/kg) during postnatal days 21-33. Starting at 70 days of age, the function and expression of receptors and transporters regulating extracellular dopamine and glutamate were determined. Juvenile PPA pretreatment completely abolished the capacity of selective dopamine and epinephrine reuptake inhibitors to increase NAC levels of both catecholamines, without impacting D2 or α2 receptor regulation of catecholamine release. Juvenile PPA pretreatment facilitated the rise in NAC glutamate elicited by dopamine, norepinephrine and glutamate transporter inhibitors and blunted mGlu2/3 inhibition of glutamate release in this region. These data confirm that juvenile exposure to PPA produces protracted perturbations in the regulation of extracellular catecholamine and glutamate levels within the NAC and further the hypothesis that early exposure to sympathomimetic drugs found in cough, cold and allergy medicines, have long-lasting effects upon neurotransmission within brain regions gating motivation.

Chronic alcohol consumption often induces hepatic steatosis but rarely causes severe inflammation in Kupffer cells (KCs) despite the increased hepatic influx of lipopolysaccharide (LPS), suggesting the presence of a veiled tolerance mechanism. In addition to LPS, the liver is affected by several gut-derived neurotransmitters through the portal blood, but the effects of catecholamines on KCs have not been clearly explored in alcohol-associated liver disease (ALD). Hence, we investigated the regulatory roles of catecholamine on inflammatory KCs under chronic alcohol exposure. We discovered that catecholamine levels were significantly elevated in the cecum, portal blood, and liver tissues of chronic ethanol-fed mice. Increased catecholamines induced mitochondrial translocation of cytochrome P450 2E1 in perivenous hepatocytes expressing the β2-adrenergic receptor (ADRB2), leading to the enhanced production of growth differentiation factor 15 (GDF15). Subsequently, GDF15 profoundly increased ADRB2 expression in adjacent inflammatory KCs to facilitate catecholamine/ADRB2-mediated apoptosis. Single-cell RNA sequencing of KCs confirmed the elevated expression of Adrb2 and apoptotic genes after chronic ethanol intake. Genetic ablation of Adrb2 or hepatic Gdf15 robustly decreased the number of apoptotic KCs near perivenous areas, exacerbating alcohol-associated inflammation. Consistently, we found that blood and stool catecholamine levels and perivenous GDF15 expression were increased in patients with early-stage ALD along with an increase in apoptotic KCs. Our findings reveal a novel protective mechanism against ALD, in which the catecholamine/GDF15 axis plays a critical role in KC apoptosis, and identify a unique neuro-metabo-immune axis between the gut and liver that elicits hepatoprotection against alcohol-mediated pathogenic challenges.

Nicotinic acetylcholine receptors (nAChRs) mediate numerous visceral functions via medullary catecholamine (CA) neurons found in the nucleus tractus solitarius (NTS), dorsal motor nucleus of the vagus (DMV), and ventrolateral medulla (VLM). However, the nAChR subtypes involved are not known. We have therefore characterized expression of nine nAChR subunit mRNAs in adult and developing rat medullary CA nuclei using combined isotopic/nonisotopic in situ hybridization. Tyrosine hydroxylase (TH) mRNA, the CA-synthesizing enzyme, was used as a marker for CA neurons, because these nuclei consist of heterogeneous populations of cells. Subunit mRNA expression varied within and between nuclei, along the rostrocaudal axis, between cell types, and across development. All CA neurons expressed beta2 mRNA, whereas alpha2 mRNA was completely absent. alpha6 And beta3 mRNA expression were restricted mainly to the VLM. alpha4, alpha5, And alpha7 mRNA expression was significantly greater in the rostral than in the caudal VLM. alpha3 And beta4 mRNAs were highly expressed in the dorsal region of the NTS, whereas dense alpha7 mRNA expression was restricted to the DMV and ventral NTS. The remaining subunit mRNAs were detected to some degree in both DMV and NTS. Except for alpha4 mRNA, which peaked prenatally, expression levels of subunit transcripts in the NTS and DMV were lower during development compared with adults. In the VLM, alpha3, alpha4, and alpha5 mRNAs expression peaked perinatally, whereas alpha6 and beta3 levels increased with age. These variations in nAChR subunit mRNA expression suggest that different receptor subtypes may produce function-specific regulation of medullary CA systems.

Activation of muscarinic receptors results in catecholamine secretion in adrenal chromaffin cells in many mammals, and muscarinic receptors partly mediate synaptic transmission from the splanchnic nerve, at least in guinea pigs. To elucidate the physiological functions of muscarinic receptors in chromaffin cells, it is necessary to identify the muscarinic receptor subtypes involved in excitation.

Comorbidity of diabetes and hypertension is frequent. Here, we have performed a comparative study in three animal models namely, normotensive Wistar Kyoto (WKY) rats, streptozotocin-induced diabetic rats (STZ), and spontaneously hypertensive rats (SHR). With respect WKY rats, we have found the following alterations in adrenal chromaffin cells from STZ and SHR rats: (1) diminished Ca2+ currents; (2) augmented [Ca2+]c elevations and catecholamine release in cells stimulated with angiotensin II or high K+; (3) unchanged expression of angiotensin II receptors AT1 and AT2; (4) higher density of secretory vesicles at subplasmalemmal sites; (5) mitochondria with lower cristae density that were partially depolarized; and (6) lower whole cell ATP content. These alterations may have their origin in (i) an augmented capacity of the endoplasmic reticulum [Ca2+] store likely due to (ii) impaired mitochondrial Ca2+ uptake; (iii) augmented high-[Ca2+]c microdomains at subplasmalemmal sites secondary to augmented calcium-induce calcium release and to inositol tris-phosphate receptor mediated enhanced Ca2+ mobilization from the endoplasmic reticulum; and (iv) augmented vesicle pool. These alterations seem to be common to the two models of human hypertension here explored, STZ diabetic rats and SHR hypertensive rats.

The A7 catecholamine cell group consists of noradrenergic (NAergic) neurons that project to the dorsal horn of the spinal cord. Here, we characterized their morphology and physiology properties and tested the effect of substance P (Sub-P) on them, since the results of many morphological studies suggest that A7 neurons are densely innervated by Sub-P-releasing terminals from nuclei involved in the descending inhibitory system, such as the lateral hypothalamus and periaqueductal gray area. Whole cell recordings were made from neurons located approximately 200 microm rostral to the trigeminal motor nucleus (the presumed A7 area) in sagittal brainstem slices from rats aged 7-10 days. After recording, the neurons were injected with biocytin and immunostained with antibody against dopamine-beta-hydroxylase (DBH). DBH-immunoreactive (ir) cells were presumed to be NAergic neurons. They had a large somata diameter ( approximately 20 microm) and relatively simple dendritic branching patterns. They fired action potentials (AP) spontaneously with or without blockade of synaptic inputs, and had similar properties to those of NAergic neurons in other areas, including the existence of calcium channel-mediated APs and a voltage-dependent delay in initiation of the AP (an indicator of the existence of A-type potassium currents) and an ability to be hyperpolarized by norepinephrine. Furthermore, in all DBH-ir neurons tested, Sub-P caused depolarization of the membrane potential and an increase in neuronal firing rate by acting on neurokinin-1 receptors. Non-DBH-ir neurons with a smaller somata size were also found in the A7 area. These showed great diversity in firing patterns and about half were depolarized by Sub-P. Morphological examination suggested that the non-DBH-ir neurons form contacts with DBH-ir neurons. These results provide the first description of the intrinsic regulation of membrane properties of, and the excitatory effect of Sub-P on, A7 area neurons, which play an important role in pain regulation.

Increasing evidence shows that the olfactory bulb is involved in blood pressure regulation in health and disease. Enhanced noradrenergic transmission in the olfactory bulb was reported in hypertension. Given that endothelins modulate catecholamines and are involved in the pathogenesis of hypertension, in the present study we sought to establish the role of the endothelin receptor type A on tyrosine hydroxylase, the rate limiting enzyme in catecholamine biosynthesis, in the olfactory bulb of DOCA-salt hypertensive rats. Sprague-Dawley male rats, randomly divided into Control and DOCA-Salt hypertensive groups, were used to assess endothelin receptors by Western blot and confocal microscopy, and their co-localization with tyrosine hydroxylase in the olfactory bulb. Blood pressure and heart rate as well as tyrosine hydroxylase expression and activity were assessed following BQ610 (ETA antagonist) applied to the brain. DOCA-Salt hypertensive rats showed enhanced ETA and decreased ETB expression. ETA co-localized with tyrosine hydroxylase positive neurons. Acute ETA blockade reduced blood pressure and heart rate and decreased the expression of total tyrosine hydroxylase and its phosphorylated forms. Furthermore, it also diminished mRNA tyrosine hydroxylase expression and accelerated the enzyme degradation through the proteasome pathway as shown by pretreatment with MG132, (20s proteasome inhibitor) intracerebroventricularly applied. Present findings support that the brain endothelinergic system plays a major role through ETA activation in the increase of catecholaminergic activity in the olfactory bulb of DOCA-Salt hypertensive rats. They provide rationale evidence that this telencephalic structure contributes in a direct or indirect way to the hemodynamic regulation in salt dependent hypertension.