Two functionally distinct alpha2-adrenergic receptors regulate sympathetic neurotransmission

The sympathetic nervous system regulates cardiovascular function by activating adrenergic receptors in the heart, blood vessels and kidney. Alpha2-adrenergic receptors are known to have a critical role in regulating neurotransmitter release from sympathetic nerves and from adrenergic neurons in the central nervous system; however, the individual roles of the three highly homologous alpha2-adrenergic-receptor subtypes (alpha2A, alpha2B, alpha2C) in this process are not known. We have now studied neurotransmitter release in mice in which the genes encoding the three alpha2-adrenergic-receptor subtypes were disrupted. Here we show that both the alpha2A- and alpha2C-subtypes are required for normal presynaptic control of transmitter release from sympathetic nerves in the heart and from central noradrenergic neurons. Alpha2A-adrenergic receptors inhibit transmitter release at high stimulation frequencies, whereas the alpha2C-subtype modulates neurotransmission at lower levels of nerve activity. Both low- and high-frequency regulation seem to be physiologically important, as mice lacking both alpha2A- and alpha2C-receptor subtypes have elevated plasma noradrenaline concentrations and develop cardiac hypertrophy with decreased left ventricular contractility by four months of age.     

Adrenergic activation of triiodothyronine production in brown adipose tissue

There are several mechanisms by which homeothermic animals increase heat production, including shivering, sympathetic nervous system activation and stimulation of thyroid hormone secretion. Studies in rats have shown that increased sympathetic activity causes increased heat production in brown adipose tissue (BAT) after cold exposure or food ingestion. Acute cold exposure also increases circulating thyroid hormones which in turn stimulate cellular metabolism through induction of various enzymes. Most metabolic effects of thyroxine (T4) are thought to be due to the triiodothyronine (T3) which is produced from T4 by a process of 5' monodeiodination. There are two enzymes responsible for this reaction: type I, or propylthiouracil (PTU)-sensitive iodothyronine deiodinase (5'D-I), which is reduced in hypothyroidism, stimulated in hyperthyroidism and probably provides most of the circulating T3 in the adult rat. Type II 5'-deiodinase (5'D-II) is characteristic of brain and pituitary, is increased by thyroidectomy, is not inhibited by PTU and provides 50-80% of the intracellular T3 in these two tissues. Recently, 5'D-II activity was identified in interscapular BAT. As the sympathetic nervous system influences the metabolic activation of BAT, we have studied the effects of noradrenaline and acute cold exposure on BAT 5'D-II. We report here that both noradrenaline and cold exposure increase BAT 5'D-II through alpha 1-adrenergic receptors, whereas depletion of catecholamines with alpha-methyl-p-tyrosine (MPT) prevents the effect of cold but not that of noradrenaline. These results suggest that the sympathetic nervous system may increase T3 production in rats by stimulating BAT 5'D-II. By increasing metabolic rate, this rise in T3 would enhance the thermogenic response to sympathetic stimulation.     

Muscarinic inhibitory transmission in mammalian sympathetic ganglia mediated by increased potassium conductance

Slow muscarinic inhibition may be a powerful influence on membrane properties in the peripheral and central nervous system. But the location of the muscarinic receptors in sympathetic ganglia, either on interneurones or on the postganglionic membrane, and the underlying mechanism of the inhibitory response, remains controversial. In mammalian sympathetic ganglia synaptic activation of muscarinic receptors located on inhibitory interneurones was thought to release catecholamines leading to a membrane hyperpolarization called the slow inhibitory postsynaptic potential, or s.-i.p.s.p.. However, the s.-i.p.s.p. in parasympathetic ganglia and in amphibian sympathetic ganglia is due to direct monosynaptic activation of muscarinic receptors, accompanied by an increased potassium conductance (but see ref. 11), and is not mediated by catecholamines. The situation is less clear in mammalian sympathetic ganglia and monosynaptic s.-i.p.s.ps observed in other ganglia could be exceptions to the hypothesis. We showed earlier that the s.-i.p.s.p. in rabbit superior cervical ganglia is not affected by catecholamine antagonists. We now show that the s.-i.p.s.p. in a mammalian sympathetic ganglion is due to the monosynaptic activation of muscarinic receptors, probably by an increase in potassium conductance.     

Identification of a factor that links apoptotic cells to phagocytes

Apoptotic cells are rapidly engulfed by phagocytes to prevent the release of potentially noxious or immunogenic intracellular materials from the dying cells, thereby preserving the integrity and function of the surrounding tissue. Phagocytes engulf apoptotic but not healthy cells, indicating that the apoptotic cells present a signal to the phagocytes, and the phagocytes recognize the signal using a specific receptor. Here, we report a factor that links apoptotic cells to phagocytes. We found that milk fat globule-EGF-factor 8 (MFG-E8), a secreted glycoprotein, was produced by thioglycollate-elicited macrophages. MFG-E8 specifically bound to apoptotic cells by recognizing aminophospholipids such as phosphatidylserine. MFG-E8, when engaged by phospholipids, bound to cells via its RGD (arginine-glycine-aspartate) motif--it bound particularly strongly to cells expressing alpha(v)beta(3) integrin. The NIH3T3 cell transformants that expressed a high level of alpha(v)beta(3) integrin were found to engulf apoptotic cells when MFG-E8 was added. MFG-E8 carrying a point mutation in the RGD motif behaved as a dominant-negative form, and inhibited the phagocytosis of apoptotic cells by peritoneal macrophages in vitro and in vivo. These results indicate that MFG-E8 secreted from activated macrophages binds to apoptotic cells, and brings them to phagocytes for engulfment.     

Resolvin E1 and protectin D1 activate inflammation-resolution programmes

Resolution of acute inflammation is an active process essential for appropriate host responses, tissue protection and the return to homeostasis. During resolution, specific omega-3 polyunsaturated fatty-acid-derived mediators are generated within resolving exudates, including resolvin E1 (RvE1) and protectin D1 (PD1). It is thus important to pinpoint specific actions of RvE1 and PD1 in regulating tissue resolution. Here we report that RvE1 and PD1 in nanogram quantities promote phagocyte removal during acute inflammation by regulating leukocyte infiltration, increasing macrophage ingestion of apoptotic polymorphonuclear neutrophils in vivo and in vitro, and enhancing the appearance of phagocytes carrying engulfed zymosan in lymph nodes and spleen. In this tissue terrain, inhibition of either cyclooxygenase or lipoxygenases--pivotal enzymes in the temporal generation of both pro-inflammatory and pro-resolving mediators--caused a 'resolution deficit' that was rescued by RvE1, PD1 or aspirin-triggered lipoxin A4 analogue. Also, new resolution routes were identified that involve phagocytes traversing perinodal adipose tissues and non-apoptotic polymorphonuclear neutrophils carrying engulfed zymosan to lymph nodes. Together, these results identify new active components for postexudate resolution traffic, and demonstrate that RvE1 and PD1 are potent agonists for resolution of inflamed tissues.     

Axoplasmic transport of muscarinic receptors

The reality of axoplasmic transport is widely accepted; various neutrotransmitters, enzymes, labelled proteins and peptides are known to move rapidly along the axons of different nerve fibres. In the terminals of sympathetic nerves, noradrenaline release is controlled by various regulatory mechanisms which imply the occurrence of presynaptic receptors. In this regard, there is considerable indirect physiological evidence for the existence of muscarinic cholinergic receptors in the sympathetic nerve endings; the stimulation by acetylcholine of such presynaptic receptors elicits an inhibitory effect on noradrenaline release. We not provide direct biochemical evidence for the occurrence in dog splenic nerve of muscarinic receptors which seem to move along the axon as suggested by their rapid accumulation on either side of a ligature.     

Alpha 1-adrenoceptor subtypes linked to different mechanisms for increasing intracellular Ca2+ in smooth muscle

Receptor-mediated increases in intracellular Ca2+ levels can be caused by release from intracellular organelles and/or influx from the extracellular fluid. Noradrenaline (NA) released from sympathetic nerves acts on alpha 1-adrenoceptors to increase cytosolic Ca2+ and promote smooth muscle contraction. In many cells activation of alpha 1-adrenoceptors causes formation of inositol 1,4,5-trisphosphate which promotes Ca2+ release from intracellular stores. The mechanism by which receptor activation opens cell surface Ca2+ channels is not known, although in some cases it may be secondary to formation of inositol phosphates or release of stored intracellular Ca2+ (ref. 3). However, alpha 1-adrenoceptors have recently been shown to have different pharmacological properties in different tissues, and it has been proposed that different alpha 1-adrenoceptor subtypes may control mobilization of intracellular Ca2+ and gating of extracellular Ca2+ influx. We here report evidence for two subtypes of alpha 1-adrenoceptors which cause contractile responses through different molecular mechanisms. One subtype stimulates inositol phosphate (InsP) formation and causes contractions which are independent of extracellular Ca2+, and the other does not stimulate inositol phosphate formation and causes contractions which require the influx of extracellular Ca2+ through dihydropyridine-sensitive channels. These results suggest that neurotransmitters and hormones may control Ca2+ release from intracellular stores and influx through voltage-gated membrane channels through distinct receptor subtypes.     

Peptidergic transmitters in synaptic boutons of sympathetic ganglia

In sympathetic ganglia of the bullfrog, a slow synaptic potential lasting for minutes--the late slow excitatory postsynaptic potential (e.p.s.p.)--was discovered. This slow response, unlike other previously known synaptic potentials in the autonomic nervous system, is not mediated by acetylcholine or monoamines. Similar non-cholinergic, non-adrenergic slow synaptic potentials have since been found in several other vertebrate autonomic ganglia. We found that the late slow e.p.s.p. is probably mediated by a peptide that is identical to, or closely resembles, mammalian luteinizing hormone releasing hormone (LHRH), because (1) when applied directly to sympathetic neurones, LHRH and its agonists elicit a slow depolarization, associated with similar changes in membrane conductance and excitability as those occurring during the late slow e.p.s.p. Furthermore, both peptide-induced and nerve-evoked responses are blocked by antagonists of LHRH; and (2) radioimmunoassays indicate that a chain of sympathetic ganglia contains 100-800 pg of a LHRH-like peptide. Its distribution among spinal nerves, the great reduction of this substance following denervation, and its release from ganglia following isotonic KCl treatment or nerve stimulation suggest that the LHRH-like material is contained in preganglionic nerve fibres. Here we report that immunohistochemical staining of sympathetic ganglia shows that LHRH-like immunoreactivity is indeed present in synaptic boutons. We also show that the two types of ganglion cells (B cells and C cells) receive strikingly different patterns of peptidergic innervation.     

Functional corticotropin releasing factor receptors in the primate peripheral sympathetic nervous system

Corticotropin releasing factor (CRF) is a key hormone in the integrated response to stress, acting both as the major regulator of pituitary adrenocorticotropic hormone (ACTH) release and as a neuropeptide in the brain. The actions of CRF are mediated by specific plasma membrane receptors in the anterior pituitary gland and in discrete brain areas including the cerebral cortex and several regions related to the limbic system. In addition to the pituitary and central actions of CRF, systemic administration of the peptide in the rat, dog, monkey and man causes hypotension and tachycardia because of a decrease in peripheral vascular resistance. These observations, in conjunction with the finding of immunoreactive and bioactive CRF in peripheral tissues, suggest that the peptide is locally released in tissues to act as a neurotransmitter or paracrine hormone. As CRF is present in the adrenal medulla and the peptide is known to modulate the central activity of the autonomic nervous system, we investigated the possibility that CRF is involved in the regulation of the peripheral autonomic nervous system. Such an action of CRF is supported by our demonstration of specific CRF receptors in the monkey adrenal medulla and sympathetic ganglia. In the adrenal medulla, these receptors are coupled to adenylate cyclase and can stimulate the secretion of catecholamines and Met-enkephalin.     

Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation

Excessive inflammation and tumour-necrosis factor (TNF) synthesis cause morbidity and mortality in diverse human diseases including endotoxaemia, sepsis, rheumatoid arthritis and inflammatory bowel disease. Highly conserved, endogenous mechanisms normally regulate the magnitude of innate immune responses and prevent excessive inflammation. The nervous system, through the vagus nerve, can inhibit significantly and rapidly the release of macrophage TNF, and attenuate systemic inflammatory responses. This physiological mechanism, termed the 'cholinergic anti-inflammatory pathway' has major implications in immunology and in therapeutics; however, the identity of the essential macrophage acetylcholine-mediated (cholinergic) receptor that responds to vagus nerve signals was previously unknown. Here we report that the nicotinic acetylcholine receptor alpha7 subunit is required for acetylcholine inhibition of macrophage TNF release. Electrical stimulation of the vagus nerve inhibits TNF synthesis in wild-type mice, but fails to inhibit TNF synthesis in alpha7-deficient mice. Thus, the nicotinic acetylcholine receptor alpha7 subunit is essential for inhibiting cytokine synthesis by the cholinergic anti-inflammatory pathway.     

RIM1alpha forms a protein scaffold for regulating neurotransmitter release at the active zone.

Neurotransmitters are released by synaptic vesicle fusion at the active zone. The active zone of a synapse mediates Ca2+-triggered neurotransmitter release, and integrates presynaptic signals in regulating this release. Much is known about the structure of active zones and synaptic vesicles, but the functional relation between their components is poorly understood. Here we show that RIM1alpha, an active zone protein that was identified as a putative effector for the synaptic vesicle protein Rab3A, interacts with several active zone molecules, including Munc13-1 (ref. 6) and alpha-liprins, to form a protein scaffold in the presynaptic nerve terminal. Abolishing the expression of RIM1alpha in mice shows that RIM1alpha is essential for maintaining normal probability of neurotransmitter release, and for regulating release during short-term synaptic plasticity. These data indicate that RIM1alpha has a central function in integrating active zone proteins and synaptic vesicles into a molecular scaffold that controls neurotransmitter release.     

Directed evolution of new catalytic activity using the alpha/beta-barrel scaffold

In biological systems, enzymes catalyse the efficient synthesis of complex molecules under benign conditions, but widespread industrial use of these biocatalysts depends crucially on the development of new enzymes with useful catalytic functions. The evolution of enzymes in biological systems often involves the acquisition of new catalytic or binding properties by an existing protein scaffold. Here we mimic this strategy using the most common fold in enzymes, the alpha/beta-barrel, as the scaffold. By combining an existing binding site for structural elements of phosphoribosylanthranilate with a catalytic template required for isomerase activity, we are able to evolve phosphoribosylanthranilate isomerase activity from the scaffold of indole-3-glycerol-phosphate synthase. We find that targeting the catalytic template for in vitro mutagenesis and recombination, followed by in vivo selection, results in a new phosphoribosylanthranilate isomerase that has catalytic properties similar to those of the natural enzyme, with an even higher specificity constant. Our demonstration of divergent evolution and the widespread occurrence of the alpha/beta-barrel suggest that this scaffold may be a fold of choice for the directed evolution of new biocatalysts.     

X-ray structure of phospholipase A2 complexed with a substrate-derived inhibitor

Phospholipases A2 play a part in a number of physiologically important cellular processes such as inflammation, blood platelet aggregation and acute hypersensitivity. These processes are all initiated by the release of arachidonic acid from cell membranes which is catalysed by intracellular phospholipases A2 and followed by conversion of arachidonic acid to prostaglandins, leukotrienes or thromboxanes. An imbalance in the production of these compounds can lead to chronic inflammatory diseases such as rheumatoid arthritis and asthma. Inhibitors of phospholipase A2 might therefore act to reduce the effects of inflammation, so structural information about the binding of phospholipase A2 to its substrates could be helpful in the design of therapeutic drugs. The three-dimensional structure is not known for any intracellular phospholipase A2, but these enzymes share significant sequence homology with secreted phospholipases, for which some of the structures have been determined. Here we report the structure of a complex between an extracellular phospholipase A2 and a competitively inhibiting substrate analogue, which reveals considerable detail about the interaction and suggests a mechanism for catalysis by this enzyme.     

Uptake of apoptotic cells drives the growth of a pathogenic trypanosome in macrophages

After apoptosis, phagocytes prevent inflammation and tissue damage by the uptake and removal of dead cells. In addition, apoptotic cells evoke an anti-inflammatory response through macrophages. We have previously shown that there is intense lymphocyte apoptosis in an experimental model of Chagas' disease, a debilitating cardiac illness caused by the protozoan Trypanosoma cruzi. Here we show that the interaction of apoptotic, but not necrotic T lymphocytes with macrophages infected with T. cruzi fuels parasite growth in a manner dependent on prostaglandins, transforming growth factor-beta (TGF-beta) and polyamine biosynthesis. We show that the vitronectin receptor is critical, in both apoptotic-cell cytoadherence and the induction of prostaglandin E2/TGF-beta release and ornithine decarboxylase activity in macrophages. A single injection of apoptotic cells in infected mice increases parasitaemia, whereas treatment with cyclooxygenase inhibitors almost completely ablates it in vivo. These results suggest that continual lymphocyte apoptosis and phagocytosis of apoptotic cells by macrophages have a role in parasite persistence in the host, and that cyclooxygenase inhibitors have potential therapeutic application in the control of parasite replication and spread in Chagas' disease.     

Beta-adrenoceptor agonists increase membrane K+ conductance in cardiac Purkinje fibres

Hormonal modulation of the ionic conductance of cell membranes is a topic of considerable current interest; it has a major role, for example, in the improved performance of the vertebrate heart elicited by sympathetic nerve stimulation or by circulating catecholamines, an effect involving enhanced calcium influx. beta-Agonist catecholamines also abbreviate the action potential of cardiac Purkinje fibres, and increase the resting potential in a variety of cells, including cardiac cells, a hyperpolarization usually attributed to stimulation of the electrogenic Na+/K+ pump. We show here that nanomolar concentrations of beta-catecholamines cause hyperpolarization of cardiac Purkinje fibres, not by increasing Na+/K+ pump current, but by increasing resting membrane K+ conductance. The hyperpolarization and shortening of the action potential should increase availability of Na+ channels and reduce the refractory period, effects tending to safeguard impulse propagation through the ventricular conducting system despite the increased heart rate caused by beta-catecholamine action on the sinus node pacemaker.     

Role for microsomal Ca storage in mammalian neurones?

Alterations in the intracellular concentration of calcium ions [( Ca2+]i) are increasingly being found to be associated with regulatory functions in cells of all kinds. In muscle, an elevation of [Ca2+]i is the final link in excitation-contraction coupling while at nerve endings and in secretory cells, similar rises in [Ca2+]i are thought to mediate exocytosis. The discovery of calcium-activated ion channels indicated a role for intracellular calcium in the regulation of membrane excitability. Calcium transients associated with either intracellular release or the inward movement of Ca2+ across the membrane have been recorded in molluscan neurons and more recently in neurones of bullfrog sympathetic ganglia. Here, we report the first recordings of calcium transients in single mammalian neurones. In these experiments we have found that the methylxanthine, caffeine, causes the release of calcium from a labile intracellular store which can be refilled by Ca2+ entering the cell during action potentials.     

Recombinant human lipocortin 1 inhibits thromboxane release from guinea-pig isolated perfused lung

The guinea-pig perfused isolated lung, used in conjunction with the cascade superfusion system to measure the release of thromboxane A2(TXA2), is a simple and convenient model for assessing the inhibition by glucocorticoids of eicosanoid formation. Dexamethasone inhibits the release of TXA2 from the lung when it is stimulated by agents such as RCS-RF2 of leukotrienes, but not when bradykinin or arachidonic acid are used. Using this model we have shown that the glucocorticoids suppress eicosanoid generation by cells through the induction of a family of phospholipase A2-inhibitory proteins now termed the 'lipocortins'. Recently the primary structure of one form of lipocortin has been elucidated and the human gene cloned. Lipocortin 1 is a polar monomeric protein with anti-phospholipase properties in vitro and we now report that when infused into guinea-pig lung preparations this protein has the same inhibitory profile as the glucocorticoids but with a more rapid onset of action. This is the first demonstration that eicosanoid formation can be inhibited by a recombinant phospholipase inhibitory protein applied extracellularly.     

Complementation in monocyte hybrids revealing genetic heterogeneity in chronic granulomatous disease

Chronic granulomatous disease (CGD) is a rare syndrome, found predominantly in male children and characterized by life-threatening, recurrent infections. The superoxide (O2-)/hydrogen peroxide (H2O2) generating system in the granulocytes and monocytes of CGD patients is completely defective. Furthermore, a novel type of cytochrome b, detected by the optical spectrum of phagocytes from healthy subjects, is lacking in those of most male CGD patients. In female CGD patients, the cytochrome b is present, but cannot, as in normal cells, be reduced on metabolic stimulation of the phagocytes in anaerobic conditions. Here, to demonstrate the importance of cytochrome b in this system and to investigate the genetic background of the various forms of CGD, we have hybridized monocytes from a cytochrome b negative, X-linked male CGD patient with monocytes from a cytochrome b positive, male CGD patient with unknown genetic background. Monocytes were used because they are the only blood phagocytes that show an active protein synthesis, whereas fibroblasts or lymphocytes do not express the O2-/H2O2 generating system. The heterologous hybrids were positive in the nitroblue tetrazolium (NBT) slide test, indicating the complementation of the O2-/H2O2 generating system, whereas the homologous hybrids remained negative, as did the non-fused cells of these patients. We thus conclude that cytochrome b is part of the O2-/H2O2 generating system and that somatic cell hybridization experiments with monocytes provide a means of studying the genetic background of CGD patients. We believe this to be the first report of genetic complementation by somatic cell hybridization experiments using monocytes instead of fibroblasts.