Adrenal serotonin derives from accumulation by the antidepressant-sensitive serotonin transporter.

Adrenal chromaffin cells comprise the neuroendocrine arm of the sympathetic nervous system and secrete catecholamines to coordinate the appropriate stress response. Deletion of the serotonin (5-HT) transporter (SERT) gene in mice (SERT-/- mice) or pharmacological block of SERT function in rodents and humans augments this sympathoadrenal stress response (epinephrine secretion). The prevailing assumption is that loss of CNS SERT alters central drive to the peripheral sympathetic nervous system. Adrenal chromaffin cells also prominently express SERT where it might coordinate accumulation of 5-HT for reuse in the autocrine control of stress-evoked catecholamine secretion. To help test this hypothesis, we have generated a novel mouse model with selective excision of SERT in the peripheral sympathetic nervous system (SERTΔTH), generated by crossing floxed SERT mice with tyrosine hydroxylase Cre driver mice. SERT expression, assessed by western blot, was abolished in the adrenal gland but not perturbed in the CNS of SERTΔTH mice. SERT-mediated [3H] 5-HT uptake was unaltered in midbrain, hindbrain, and spinal cord synaptosomes, confirming transporter function was intact in the CNS. Endogenous midbrain and whole blood 5-HT homeostasis was unperturbed in SERTΔTH mice, contrasting with the depleted 5-HT content in SERT-/- mice. Selective SERT excision reduced adrenal gland 5-HT content by ≈ 50% in SERTΔTH mice but had no effect on adrenal catecholamine content. This novel model confirms that SERT expressed in adrenal chromaffin cells is essential for maintaining wild-type levels of 5-HT and provides a powerful tool to help dissect the role of SERT in the sympathetic stress response.

Sigma-1 receptor ligands inhibit catecholamine secretion from adrenal chromaffin cells due to block of nicotinic acetylcholine receptors.

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.

Serotonin and the serotonin transporter in the adrenal gland.

The adrenal glands are key components of the mammalian endocrine system, helping maintain physiological homeostasis and the coordinated response to stress. Each adrenal gland has two morphologically and functionally distinct regions, the outer cortex and inner medulla. The cortex is organized into three concentric zones which secrete steroid hormones, including aldosterone and cortisol. Neural crest-derived chromaffin cells in the medulla are innervated by preganglionic sympathetic neurons and secrete catecholamines (epinephrine, norepinephrine) and neuropeptides into the bloodstream, thereby functioning as the neuroendocrine arm of the sympathetic nervous system. In this article we review serotonin (5-HT) and the serotonin transporter (SERT; SLC6A4) in the adrenal gland. In the adrenal cortex, 5-HT, primarily sourced from resident mast cells, acts as a paracrine signal to stimulate aldosterone and cortisol secretion through 5-HT4/5-HT7 receptors. Medullary chromaffin cells contain a small amount of 5-HT due to SERT-mediated uptake and express 5-HT1A receptors which inhibit secretion. The atypical mechanism of the 5-HT1A receptors and interaction with SERT fine tune this autocrine pathway to control stress-evoked catecholamine secretion. Receptor-independent signaling by SERT/intracellular 5-HT modulates the amount and kinetics of transmitter release from single vesicle fusion events. SERT might also influence stress-evoked upregulation of tyrosine hydroxylase transcription. Transient signaling via 5-HT3 receptors during embryonic development can limit the number of chromaffin cells found in the mature adrenal gland. Together, this emerging evidence suggests that the adrenal medulla is a peripheral hub for serotonergic control of the sympathoadrenal stress response.

Jedi-1 deficiency increases sensory neuron excitability through a non-cell autonomous mechanism.

The dorsal root ganglia (DRG) house the primary afferent neurons responsible for somatosensation, including pain. We previously identified Jedi-1 (PEAR1/MEGF12) as a phagocytic receptor expressed by satellite glia in the DRG involved in clearing apoptotic neurons during development. Here, we further investigated the function of this receptor in vivo using Jedi-1 null mice. In addition to satellite glia, we found Jedi-1 expression in perineurial glia and endothelial cells, but not in sensory neurons. We did not detect any morphological or functional changes in the glial cells or vasculature of Jedi-1 knockout mice. Surprisingly, we did observe changes in DRG neuron activity. In neurons from Jedi-1 knockout (KO) mice, there was an increase in the fraction of capsaicin-sensitive cells relative to wild type (WT) controls. Patch-clamp electrophysiology revealed an increase in excitability, with a shift from phasic to tonic action potential firing patterns in KO neurons. We also found alterations in the properties of voltage-gated sodium channel currents in Jedi-1 null neurons. These results provide new insight into the expression pattern of Jedi-1 in the peripheral nervous system and indicate that loss of Jedi-1 alters DRG neuron activity indirectly through an intercellular interaction between non-neuronal cells and sensory neurons.

Serotonin and Serotonin Transporters in the Adrenal Medulla: A Potential Hub for Modulation of the Sympathetic Stress Response.

Serotonin (5-HT) is an important neurotransmitter in the central nervous system where it modulates circuits involved in mood, cognition, movement, arousal, and autonomic function. The 5-HT transporter (SERT; SLC6A4) is a key regulator of 5-HT signaling, and genetic variations in SERT are associated with various disorders including depression, anxiety, and autism. This review focuses on the role of SERT in the sympathetic nervous system. Autonomic/sympathetic dysfunction is evident in patients with depression, anxiety, and other diseases linked to serotonergic signaling. Experimentally, loss of SERT function (SERT knockout mice or chronic pharmacological block) has been reported to augment the sympathetic stress response. Alterations to serotonergic signaling in the CNS and thus central drive to the peripheral sympathetic nervous system are presumed to underlie this augmentation. Although less widely recognized, SERT is robustly expressed in chromaffin cells of the adrenal medulla, the neuroendocrine arm of the sympathetic nervous system. Adrenal chromaffin cells do not synthesize 5-HT but accumulate small amounts by SERT-mediated uptake. Recent evidence demonstrated that 5-HT1A receptors inhibit catecholamine secretion from adrenal chromaffin cells via an atypical mechanism that does not involve modulation of cellular excitability or voltage-gated Ca2+ channels. This raises the possibility that the adrenal medulla is a previously unrecognized peripheral hub for serotonergic control of the sympathetic stress response. As a framework for future investigation, a model is proposed in which stress-evoked adrenal catecholamine secretion is fine-tuned by SERT-modulated autocrine 5-HT signaling.

Modifications of the human urocortin 2 peptide that improve pharmacological properties.

Recently, we demonstrated that the corticotropin releasing factor 2 receptor agonist, urocortin 2, demonstrated anti-atrophy effects in rodent skeletal muscle atrophy models. Compared to other CRF2R agonists however, the in vivo pharmacological potency of urocortin 2 is poor when it is administered by continuous subcutaneous infusion. Therefore, we attempted to modify the structure of urocortin 2 to improve in vivo efficacy when administered by subcutaneous infusion. By substituting amino acid residues in the linker region of urocortin 2 (residues 22-32), we have demonstrated improved in vivo potency without altering selectivity, probably through reduced CRFBP binding. In addition, attempts to shorten urocortin 2 generally resulted in inactive peptides, demonstrating that the 38 amino acid urocortin 2 peptide is the minimal pharmacophore.

An interplay between the serotonin transporter (SERT) and 5-HT receptors controls stimulus-secretion coupling in sympathoadrenal chromaffin cells.

Adrenal chromaffin cells (ACCs), the neuroendocrine arm of the sympathetic nervous system, secrete catecholamines to mediate the physiological response to stress. Although ACCs do not synthesize 5-HT, they express the serotonin transporter (SERT). Genetic variations in SERT are linked to several CNS disorders but the role(s) of SERT/5-HT in ACCs has remained unclear. Adrenal glands from wild-type mice contained 5-HT at ≈ 750 fold lower abundance than adrenaline, and in SERT(-/-) mice this was reduced by ≈80% with no change in catecholamines. Carbon fibre amperometry showed that SERT modulated the ability of 5-HT1A receptors to inhibit exocytosis. 5-HT reduced the number of amperometric spikes (vesicular fusion events) evoked by KCl in SERT(-/-) cells and wild-type cells treated with escitalopram, a SERT antagonist. The 5-HT1A receptor antagonist WAY100635 blocked the inhibition by 5-HT which was mimicked by the 5-HT1A agonist 8-OH-DPAT but not the 5-HT1B agonist CP93129. There was no effect on voltage-gated Ca(2+) channels, K(+) channels, or intracellular [Ca(2+)] handling, showing the 5-HT receptors recruit an atypical inhibitory mechanism. Spike charge and kinetics were not altered by 5-HT receptors but were reduced in SERT(-/-) cells compared to wild-type cells. Our data reveal a novel role for SERT and suggest that adrenal chromaffin cells might be a previously unrecognized hub for serotonergic control of the sympathetic stress response.

Discovery of corticotropin releasing factor 2 receptor selective sauvagine analogues for treatment of skeletal muscle atrophy.

The corticotropin release factor 2 receptor (CRF2R) has many biological activities including modulation of the stress response. Recently, we have demonstrated that CRF2R activation functions to prevent skeletal muscle wasting resulting from a variety of physiological stimuli. Thus we are interested in identifying CRF2R selective agonists with optimal pharmacological properties for use in treating muscle wasting diseases. Several CRF2R agonists are known including the frog peptide sauvagine (Svg), which display superior pharmacological properties compared to other CRF2R agonists. Unfortunately sauvagine is a nonselective CRFR agonist, thus making it of less utility due to side effects resulting from corticotropin release factor 1 receptor (CRF1R) activation. Because our initial modifications of Svg at position 11 improved CRF2R selectivity, we investigated the role of amino acids at positions 12 and 13 in Svg. We observed that phenylalanine, leucine, isoleucine, threonine, glutamine, histidine, and tyrosine at the 12th position were the strongest promoters of CRF2R selectivity whereas phenylalanine, glutamine, trytophane, tyrosine, valine, isoleucine, leucine, and 2-naphthylalanine were the preferred residues at the 13th position. Selective sauvagine peptides demonstrated improved antiatrophy effects in a mouse-casting model when compared to sauvagine itself. Thus, we demonstrate that the CRF2R selectivity can be improved by optimizing amino acids at positions 12 and 13 (all with proline at position 11) and that the selective sauvagine analogues demonstrate better in vivo efficacy than sauvagine itself.

Urocortin II treatment reduces skeletal muscle mass and function loss during atrophy and increases nonatrophying skeletal muscle mass and function.

Two corticotropin-releasing factor 2 receptor (CRF2R)-selective peptides have been recently described, urocortin II (also known as stresscopin-related peptide) and urocortin III (stresscopin). We have used urocortin II to evaluate the effects of activation of the CRF2R on skeletal muscle-related physiological processes. Administration of urocortin II to mice prevented the loss of skeletal muscle mass resulting from disuse due to casting, corticosteroid treatment, and nerve damage. In addition, urocortin II treatment prevented the loss of skeletal muscle force and myocyte cross-sectional area that accompanied muscle mass losses resulting from disuse due to casting. Finally, we observed increased skeletal muscle mass and force in normal muscles when mice are treated with urocortin II. These results were confirmed using two additional CRF2R agonists, urocortin I and sauvagine. Thus, activation of the CRF2R modulates skeletal muscle mass in both normal and atrophying muscle. Therefore, CRF2R-selective agonists may find utility in the treatment of skeletal muscle wasting diseases including age-related muscle loss or sarcopenia.

Butanol isomers exert distinct effects on voltage-gated calcium channel currents and thus catecholamine secretion in adrenal chromaffin cells.

Butanol (C4H10OH) has been used both to dissect the molecular targets of alcohols/general anesthetics and to implicate phospholipase D (PLD) signaling in a variety of cellular functions including neurotransmitter and hormone exocytosis. Like other primary alcohols, 1-butanol is a substrate for PLD and thereby disrupts formation of the intracellular signaling lipid phosphatidic acid. Because secondary and tertiary butanols do not undergo this transphosphatidylation, they have been used as controls for 1-butanol to implicate PLD signaling. Recently, selective pharmacological inhibitors of PLD have been developed and, in some cases, fail to block cellular functions previously ascribed to PLD using primary alcohols. For example, exocytosis of insulin and degranulation of mast cells are blocked by primary alcohols, but not by the PLD inhibitor FIPI. In this study we show that 1-butanol reduces catecholamine secretion from adrenal chromaffin cells to a much greater extent than tert-butanol, and that the PLD inhibitor VU0155056 has no effect. Using fluorescent imaging we show the effect of these drugs on depolarization-evoked calcium entry parallel those on secretion. Patch-clamp electrophysiology confirmed the peak amplitude of voltage-gated calcium channel currents (I(Ca)) is inhibited by 1-butanol, with little or no block by secondary or tert-butanol. Detailed comparison shows for the first time that the different butanol isomers exert distinct, and sometimes opposing, effects on the voltage-dependence and gating kinetics of I(Ca). We discuss these data with regard to PLD signaling in cellular physiology and the molecular targets of general anesthetics.

Phosphodiesterase 4 inhibition reduces skeletal muscle atrophy.

Several GTP-binding protein (G-protein)-coupled receptors that signal through Galphas (GTP-binding protein alpha stimulatory) and the cyclic adenosine monophosphate (cAMP) pathway increase skeletal muscle mass. In order to further evaluate the role of the cAMP pathway in the regulation of skeletal muscle mass, we utilized inhibitors of phosphodiesterase 4 (PDE 4), the major cAMP-modifying PDE found in skeletal muscle, to modulate skeletal muscle cAMP levels. We found that PDE 4 inhibitors reduced the loss of muscle mass and force resulting from denervation and casting in rats and mice. These studies indicate that PDE 4 inhibitors may have a role in the treatment of skeletal muscle-wasting diseases.

Interacting effects of N-terminal variation and strex exon splicing on slo potassium channel regulation by calcium, phosphorylation, and oxidation.

We have investigated the structural basis for the phenotype of a native rat Slo (rSlo) potassium channel (BK(Ca); KCNMA1) in a rat pituitary cell line, GH(4)C(1). Opposing regulation of these calcium- and voltage-activated potassium channels by cAMP- and cGMP-dependent protein kinases requires an alternatively spliced exon (strex) of 59 amino acids in the cytoplasmic C terminus of the pore-forming alpha subunit encoded by rslo. However, inclusion of this cysteine-rich exon produces a 10-fold increase in the sensitivity of the channels to inhibition by oxidation. Inclusion of the strex exon also increases channel sensitivity to stimulation by calcium, but responses in the physiological ranges of calcium and voltage require coassembly with beta(1) subunits. With strex present, however, beta(1) subunits only stimulated channels assembled from rSlo alpha subunits with a truncated N terminus beginning MDALI-. Thus N-terminal variation and strex exon splicing in rSlo interact to produce BK(Ca) channels with a physiologically relevant phenotype.

Activation of the CRF 2 receptor modulates skeletal muscle mass under physiological and pathological conditions.

Two receptors activated by the corticotropin-releasing factor (CRF) family of peptides have been identified, the CRF 1 receptor (CRF1R) and the CRF 2 receptor (CRF2R). Of these, the CRF2R is expressed in skeletal muscle. To understand the role of the CRF2R in skeletal muscle, we utilized CRFR knockout mice and CRF2R-selective agonists to modulate nerve damage and corticosteroid- and disuse-induced skeletal muscle atrophy in mice. These analyses demonstrated that activation of the CRF2R decreased nerve damage and corticosteroid- and disuse-induced skeletal muscle mass and function loss. In addition, selective activation of the CRF2R increased nonatrophy skeletal muscle mass. Thus we describe for the first time a novel activity of the CRF2R, modulation of skeletal muscle mass.