Conversion of agonist site to metal-ion chelator site in the β2-adrenergic receptor

Previously metal-ion sites have been used as structural and functional probes in seven transmembrane receptors (7TM), but as yet all the engineered sites have been inactivating. Based on presumed agonist interaction points in transmembrane III (TM-III) and -VII of the β2-adrenergic receptor, in this paper we construct an activating metal-ion site between the amine-binding Asp-113 in TM-III—or a His residue introduced at this position—and a Cys residue substituted for Asn-312 in TM-VII. No increase in constitutive activity was observed in the mutant receptors. Signal transduction was activated in the mutant receptors not by normal catecholamine ligands but instead either by free zinc ions or by zinc or copper ions in complex with small hydrophobic metal-ion chelators. Chelation of the metal ions by small hydrophobic chelators such as phenanthroline or bipyridine protected the cells from the toxic effect of, for example Cu2+, and in several cases increased the affinity of the ions for the agonistic site. Wash-out experiments and structure–activity analysis indicated, that the high-affinity chelators and the metal ions bind and activate the mutant receptor as metal ion guided ligand complexes. Because of the well-understood binding geometry of the small metal ions, an important distance constraint has here been imposed between TM-III and -VII in the active, signaling conformation of 7TM receptors. It is suggested that atoxic metal-ion chelator complexes could possibly in the future be used as generic, pharmacologic tools to switch 7TM receptors with engineered metal-ion sites on or off at will.

Protein kinase C as a signal for exocytosis

We have studied signaling mechanisms that stimulate exocytosis and luteinizing hormone secretion in isolated male rat pituitary gonadotropes. As judged by reverse hemolytic plaque assays, phorbol-12-myristate-13-acetate (PMA) stimulates as many gonadotropes to secrete as does gonadotropin-releasing hormone (GnRH). However, PMA and GnRH use different signaling pathways. The secretagogue action of GnRH is not very sensitive to bisindolylmaleimide I, an inhibitor of protein kinase C, but is blocked by loading cells with a calcium chelator, 1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. The secretagogue action of PMA is blocked by bisindolylmaleimide I and is not very sensitive to the intracellular calcium chelator. GnRH induces intracellular calcium elevations, whereas PMA does not. As judged by amperometric measurements of quantal catecholamine secretion from dopamine- or serotonin-loaded gonadotropes, the secretagogue action of PMA develops more slowly (in several minutes) than that of GnRH. We conclude that exocytosis of secretory vesicles can be stimulated independently either by calcium elevations or by activation of protein kinase C.

Dopamine β-hydroxylase deficiency impairs cellular immunity

Norepinephrine, released from sympathetic neurons, and epinephrine, released from the adrenal medulla, participate in a number of physiological processes including those that facilitate adaptation to stressful conditions. The thymus, spleen, and lymph nodes are richly innervated by the sympathetic nervous system, and catecholamines are thought to modulate the immune response. However, the importance of this modulatory role in vivo remains uncertain. We addressed this question genetically by using mice that lack dopamine β-hydroxylase (dbh−/− mice). dbh−/− mice cannot produce norepinephrine or epinephrine, but produce dopamine instead. When housed in specific pathogen-free conditions, dbh−/− mice had normal numbers of blood leukocytes, and normal T and B cell development and in vitro function. However, when challenged in vivo by infection with the intracellular pathogens Listeria monocytogenes or Mycobacterium tuberculosis, dbh−/− mice were more susceptible to infection, exhibited extreme thymic involution, and had impaired T cell function, including Th1 cytokine production. When immunized with trinitrophenyl-keyhole limpet hemocyanin, dbh−/− mice produced less Th1 cytokine-dependent-IgG2a antitrinitrophenyl antibody. These results indicate that physiological catecholamine production is not required for normal development of the immune system, but plays an important role in the modulation of T cell-mediated immunity to infection and immunization.

Loss of signaling through the G protein, Gz, results in abnormal platelet activation and altered responses to psychoactive drugs

Heterotrimeric G proteins mediate the earliest step in cell responses to external events by linking cell surface receptors to intracellular signaling pathways. Gz is a member of the Gi family of G proteins that is prominently expressed in platelets and brain. Here, we show that deletion of the α subunit of Gz in mice: (i) impairs platelet aggregation by preventing the inhibition of cAMP formation normally seen at physiologic concentrations of epinephrine, and (ii) causes the mice to be more resistant to fatal thromboembolism. Loss of Gzα also results in greatly exaggerated responses to cocaine, reduces the analgesic effects of morphine, and abolishes the effects of widely used antidepressant drugs that act as catecholamine reuptake inhibitors. These changes occur despite the presence of other Giα family members in the same cells and are not accompanied by detectable compensatory changes in the level of expression of other G protein subunits. Therefore, these results provide insights into receptor selectivity among G proteins and a model for understanding platelet function and the effects of psychoactive drugs.

Oocytes are a source of catecholamines in the primate ovary: Evidence for a cell–cell regulatory loop

Catecholamines, thought to derive from the extrinsic innervation of the ovary, participate in the regulation of ovarian development and mature gonadal function. Recently, intraovarian neurons containing tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, were described in the ovary of nonhuman primates. We now show that the primate ovary expresses both the genes encoding TH and dopamine β-hydroxylase (DBH), the key enzymes in norepinephrine (NE) biosynthesis. Ovarian neurons were identified as a site of TH and DBH gene expression, and surprisingly, oocytes were identified as an exclusive site of DBH synthesis. Oocytes contain neither TH mRNA nor protein, indicating that they are unable to synthesize dopamine (DA). They did, however, express a DA transporter gene identical to that found in human brain. The physiological relevance of this transporter system and DBH in oocytes was indicated by the ability of isolated oocytes to metabolize exogenous DA into NE. Isolated follicles containing oocytes—but not those from which the oocytes had been removed—responded to DA with an elevation in cAMP levels; this elevation was prevented by propranolol, a β-adrenoreceptor antagonist. The results suggest that oocytes and somatic cells are linked by a neuroendocrine loop consisting of NE synthesized in oocytes from actively transported DA and cAMP produced by somatic follicular cells in response to NE-induced β-adrenoreceptor activation.

Targeted inhibition of calcineurin attenuates cardiac hypertrophy in vivo

The Ca2+-calmodulin-activated Ser/Thr protein phosphatase calcineurin and the downstream transcriptional effectors of calcineurin, nuclear factor of activated T cells, have been implicated in the hypertrophic response of the myocardium. Recently, the calcineurin inhibitory agents cyclosporine A and FK506 have been extensively used to evaluate the importance of this signaling pathway in rodent models of cardiac hypertrophy. However, pharmacologic approaches have rendered equivocal results necessitating more specific or genetic-based inhibitory strategies. In this regard, we have generated Tg mice expressing the calcineurin inhibitory domains of Cain/Cabin-1 and A-kinase anchoring protein 79 specifically in the heart. ΔCain and ΔA-kinase-anchoring protein Tg mice demonstrated reduced cardiac calcineurin activity and reduced hypertrophy in response to catecholamine infusion or pressure overload. In a second approach, adenoviral-mediated gene transfer of ΔCain was performed in the adult rat myocardium to evaluate the effectiveness of an acute intervention and any potential species dependency. ΔCain adenoviral gene transfer inhibited cardiac calcineurin activity and reduced hypertrophy in response to pressure overload without reducing aortic pressure. These results provide genetic evidence implicating calcineurin as an important mediator of the cardiac hypertrophic response in vivo.

Myocardial signaling defects and impaired cardiac function of a human beta 2-adrenergic receptor polymorphism expressed in transgenic mice.

A threonine to isoleucine polymorphism at amino acid 164 in the fourth transmembrane spanning domain of the beta 2-adrenergic receptor (beta 2AR) is known to occur in the human population. The functional consequences of this polymorphism to catecholamine signaling in relevant cells or to end-organ responsiveness, however, are not known. To explore potential differences between the two receptors, site-directed mutagenesis was carried out to mimic the polymorphism. Transgenic FVB/N mice were then created overexpressing wild-type (wt) beta 2AR or the mutant Ile-164 receptor in a targeted manner in the heart using a murine alpha myosin heavy chain promoter. The functional properties of the two receptors were then assessed at the level of in vitro cardiac myocyte signaling and in vivo cardiac responses in intact animals. The expression levels of these receptors in the two lines chosen for study were approximately 1200 fmol/mg protein in cardiac membranes, which represents a approximately 45-fold increase in expression over endogenous beta AR. Myocyte membrane adenylyl cyclase activity in the basal state was significantly lower in the Ile-164 mice (19.5 +/- 2.7 pmol/min/mg) compared with wt beta 2AR mice (35.0 +/- 4.1 pmol/min/mg), as was the maximal isoproterenol-stimulated activity (49.8 +/- 7.8 versus 77.1 +/ 7.3 pmol/min/mg). In intact animals, resting heart rate (441 +/- 21 versus 534 +/- 17 bpm) and dP/dtmax (10,923 +/- 730 versus 15,308 +/- 471 mmHg/sec) were less in the Ile-164 mice as compared with wt beta 2AR mice. Similarly, the physiologic responses to infused isoproterenol were notably less in the mutant expressing mice. Indeed, these values, as well as other contractile parameters, were indistinguishable between Ile-164 mice and nontransgenic littermates. Taken together, these results demonstrate that the Ile-164 polymorphism is substantially dysfunctional in a relevant target tissue, as indicated by depressed receptor coupling to adenylyl cyclase in myocardial membranes and impaired receptor mediated cardiac function in vivo. Under normal homeostatic conditions or in circumstances where sympathetic responses are compromised due to diseased states, such as heart failure, this impairment may have important pathophysiologic consequences.

Olfactory marker protein (OMP) gene deletion causes altered physiological activity of olfactory sensory neurons.

Olfactory marker protein (OMP) is an abundant, phylogentically conserved, cytoplasmic protein of unknown function expressed almost exclusively in mature olfactory sensory neurons. To address its function, we generated OMP-deficient mice by gene targeting in embryonic stem cells. We report that these OMP-null mice are compromised in their ability to respond to odor stimull, providing insight to OMP function. The maximal electroolfactogram response of the olfactory neuroepithelium to several odorants was 20-40% smaller in the mutants compared with controls. In addition, the onset and recovery kinetics following isoamyl acetate stimulation are prolonged in the null mice. Furthermore, the ability of the mutants to respond to the second odor pulse of a pair is impaired, over a range of concentrations, compared with controls. These results imply that neural activity directed toward the olfactory bulb is also reduced. The bulbar phenotype observed in the OMP-null mouse is consistent with this hypothesis. Bulbar activity of tyrosine hydroxylase, the rate limiting enzyme of catecholamine biosynthesis, and content of the neuropeptide cholecystokinin are reduced by 65% and 50%, respectively. This similarity to postsynaptic changes in gene expression induced by peripheral olfactory deafferentation or naris blockade confirms that functional neural activity is reduced in both the olfactory neuroepithelium and the olfactory nerve projection to the bulb in the OMP-null mouse. These observations provide strong support for the conclusion that OMP is a novel modulatory component of the odor detection/signal transduction cascade.

G protein subunits and the stimulation of phospholipase C by Gs-and Gi-coupled receptors: Lack of receptor selectivity of Galpha(16) and evidence for a synergic interaction between Gbeta gamma and the alpha subunit of a receptor activated G protein.

Stimulatory guanine nucleotide binding protein (Gs)-coupled receptors activated by luteinizing hormone, vasopressin, and the catecholamine isoproterenol (luteinizing hormone receptor, type 2 vasopressin receptor, and types 1 and 2 beta-adrenergic receptors) and the Gi-coupled M2 muscarinic receptor (M2R) were expressed transiently in COS cells, alone and in combination with Gbeta gamma dimers, their corresponding Galphas (Galpha(s), or Galpha(i3)) and either Galpha(q) or Galpha(16). Phospholipase C (PLC) activity, assessed by inositol phosphate production from preincorporated myo[3H]inositol, was then determined to gain insight into differential coupling preferences among receptors and G proteins. The following were observed: (i) All receptors tested were able to stimulate PLC activity in response to agonist occupation. The effect of the M2R was pertussis toxin sensitive. (ii) While, as expected, expression of Galpha(q) facilitated an agonist-induced activation of PLC that varied widely from receptor to receptor (400% with type 2 vasopressin receptor and only 30% with M2R), expression of Galpha(16) facilitated about equally well the activation of PLC by any of the tested receptors and thus showed little if any discrimination for one receptor over another. (iii) Gbeta gamma elevated basal (agonist independent) PLC activity between 2- and 4-fold, confirming the proven ability of Gbeta gamma to stimulate PLCbeta. (iv) Activation of expressed receptors by their respective ligands in cells coexpressing excess Gbeta gamma elicited agonist stimulated PLC activities, which, in the case of the M2R, was not blocked by pertussis toxin (PTX), suggesting mediation by a PTX-insensitive PLC-stimulating Galpha subunit, presumably, but not necessarily, of the Gq family. (v) The effects of Gbeta gamma and the PTX-insensitive Galpha elicited by M2R were synergistic, suggesting the possibility that one or more forms of PLC are under conditional or dual regulation of G protein subunits such that stimulation by one sensitizes to the stimulation by the other.

Olfactory neuroblastoma is a peripheral primitive neuroectodermal tumor related to Ewing sarcoma.

Olfactory neuroblastoma (ONB) is a malignant tumor of the nasal mucosa whose histogenesis is unclear. A relationship to neuroblastoma (NB), a pediatric tumor of the sympathetic nervous system, is based on morphologic similarities and the expression of similar neural antigens. However, the clinical presentation of ONB differs from that of NB, and MYCN amplification characteristic of NB is not observed. We have therefore examined the relationship of this malignancy to other classes of neural tumors. In previous studies, two ONB cell lines demonstrated cytogenetic features and patterns of protooncogene expression suggestive of a relationship to the Ewing sarcoma family of childhood peripheral primitive neuroectodermal tumors (pPNETs). The pPNETs show t(11;22)(q24;q12) or t(21;22)(q22;q12) chromosomal translocations fusing the EWS gene from 22q12 with either the FL11 gene on 11q24 or the ERG gene on 21q22. We therefore analyzed ONBs for the presence of pPNET-associated gene fusions. Both cell lines showed rearrangement of the EWS gene, and fluorescence in situ hybridization (FISH) of each case demonstrated fusion of EWS and FL11 genomic sequences. Moreover, both lines expressed EWS/FL11 fusion transcripts with in-frame junctions between exon 7 of EWS and exon 6 of FL11 as described for pPNETs. We identified similar gene fusions in four of six primary ONB cases. None of the cases expressed tyrosine hydroxylase, a catecholamine biosynthetic enzyme widely expressed in NB. Our studies indicate that ONB is not a NB but is a member of the pPNET family.

Neurosecretory vesicles can be hybrids of synaptic vesicles and secretory granules.

We have investigated the relationship of the so-called small dense core vesicle (SDCV), the major catecholamine-containing neurosecretory vesicle of sympathetic neurons, to synaptic vesicles containing classic neurotransmitters and secretory granules containing neuropeptides. SDCVs contain membrane proteins characteristic of synaptic vesicles such as synaptophysin and synaptoporin. However, SDCVs also contain membrane proteins characteristic of certain secretory granules like the vesicular monoamine transporter and the membrane-bound form of dopamine beta-hydroxylase. In neurites of sympathetic neurons, synaptophysin and dopamine beta-hydroxylase are found in distinct vesicles, consistent with their transport from the trans-Golgi network to the site of SDCV formation in constitutive secretory vesicles and secretory granules, respectively. Hence, SDCVs constitute a distinct type of neurosecretory vesicle that is a hybrid of the synaptic vesicle and the secretory granule membranes and that originates from the contribution of both the constitutive and the regulated pathway of protein secretion.

Amperometric detection of stimulus-induced quantal release of catecholamines from cultured superior cervical ganglion neurons.

Amperometry has been used for real-time electrochemical detection of the quantal release of catecholamines and indolamines from secretory granules in chromaffin and mast cells. Using improved-sensitivity carbon fiber electrodes, we now report the detection of quantal catecholamine release at the surface of somas of neonatal superior cervical ganglion neurons that are studded with axon varicosities containing synaptic vesicles. Local application of a bath solution containing high K+ or black widow spider venom, each of which greatly enhances spontaneous quantal release of transmitter at synapses, evoked barrages of small-amplitude (2-20 pA), short-duration (0.5-2 ms) amperometric quantal "spikes". The median spike charge was calculated as 11.3 fC. This figure corresponds to 3.5 x 10(4) catecholamine molecules per quantum of release, or approximately 1% that evoked by the discharge of the contents of a chromaffin granule.

Colocalization of calcium entry and exocytotic release sites in adrenal chromaffin cells.

"Snapshot" images of localized Ca2+ influx into patch-clamped chromaffin cells were captured by using a recently developed pulsed-laser imaging system. Transient opening of voltage-sensitive Ca2+ channels gave rise to localized elevations of Ca2+ that had the appearance of either "hotspots" or partial rings found immediately beneath the plasma membrane. When the Ca2+ imaging technique was employed in conjunction with flame-etched carbon-fiber electrodes to spatially map the release sites of catecholamines, it was observed that the sites of Ca2+ entry and catecholamine release were colocalized. These results provide functional support for the idea that secretion occurs from "active zone"-like structures in neuroendocrine cells.

Propofol infusion syndrome in a super morbidly obese patient (BMI = 75)

Propofol infusion syndrome (PRIS) is a rare but often fatal complication as a result of large doses of propofol infusion (4–5 mg/kg/hr) for a prolonged period (>48 h). It has been reported in both children and adults. Besides large doses of propofol infusion, the risk factors include young age, acute neurological injury, low carbohydrate and high fat intake, exogenous administration of corticosteroid and catecholamine, critical illness, and inborn errors of mitochondrial fatty acid oxidation. PRIS manifestation include presence of metabolic acidosis with a base deficit of more than 10 mmol/l at least on one occasion, rhabdomyolysis or myoglobinuria, acute renal failure, sudden onset of bradycardia resistant to treatment, myocardial failure, and lipemic plasma. The pathophysiology of PRIS may be either direct mitochondrial respiratory chain inhibition or impaired mitochondrial fatty acid metabolism mediated by propofol. We report a case of supermorbidly obese patient who received propofol infusion by total body weight instead of actual body weight and developed PRIS.

Fatigue and Co-occurring Symptoms in Women with Irritable Bowel Syndrome

Thesis (Ph.D.)--University of Washington, 2016-06