AB5986 Sigma-AldrichAnti-Tyrosine Hydroxylase Antibody

Changes in airway nerves associated with chronic inflammation may underlie the pathogenesis and symptoms of lower airway diseases, such as asthma. The molecules most likely causing such alterations are neurotrophins (NTs) and/or related neurokines. In several species, including humans, lower airway parasympathetic postganglionic neurons that project axons to airway smooth muscle are either cholinergic or nonadrenergic noncholinergic (NANC), the latter synthesizing vasoactive intestinal peptide and nitric oxide, but not acetylcholine. In guinea pig trachealis smooth muscle, cholinergic nerve terminals arise from ganglionic neurons located near the tracheal smooth muscle, whereas the source of NANC nerve fibers is from neurons in ganglia located in the adjacent myenteric plexus of the esophagus, making this an ideal species to study regulation of parasympathetic neurotransmitter phenotypes. In the present study, we determined that, 48 hours after repeated allergen challenge, the NANC phenotype of airway parasympathetic ganglionic neurons changed to a cholinergic phenotype, and NT-3 mimicked this change. Nerve growth factor, brain-derived neurotrophic factor, leukemia inhibitory factor, or IL-1β had no effect on either phenotype, and they did not induce these neurons to synthesize substance P or tyrosine hydroxylase. These results indicate a role for inflammation and NT-3 in regulating biochemical and anatomical characteristics of principal neurons in adult airway parasympathetic ganglia.

Recent experiments suggest that brainstem GABAergic neurons may control rapid-eye-movement (REM) sleep. However, understanding their pharmacology/physiology has been hindered by difficulty in identification. Here we report that mice expressing green fluorescent protein (GFP) under the control of the GAD67 promoter (GAD67-GFP knock-in mice) exhibit numerous GFP-positive neurons in the central gray and reticular formation, allowing on-line identification in vitro. Small (10-15 microm) or medium-sized (15-25 microm) GFP-positive perikarya surrounded larger serotonergic, noradrenergic, cholinergic and reticular neurons, and > 96% of neurons were double-labeled for GFP and GABA, confirming that GFP-positive neurons are GABAergic. Whole-cell recordings in brainstem regions important for promoting REM sleep [subcoeruleus (SubC) or pontine nucleus oralis (PnO) regions] revealed that GFP-positive neurons were spontaneously active at 3-12 Hz, fired tonically, and possessed a medium-sized depolarizing sag during hyperpolarizing steps. Many neurons also exhibited a small, low-threshold calcium spike. GFP-positive neurons were tested with pharmacological agents known to promote (carbachol) or inhibit (orexin A) REM sleep. SubC GFP-positive neurons were excited by the cholinergic agonist carbachol, whereas those in the PnO were either inhibited or excited. GFP-positive neurons in both areas were excited by orexins/hypocretins. These data are congruent with the hypothesis that carbachol-inhibited GABAergic PnO neurons project to, and inhibit, REM-on SubC reticular neurons during waking, whereas carbachol-excited SubC and PnO GABAergic neurons are involved in silencing locus coeruleus and dorsal raphe aminergic neurons during REM sleep. Orexinergic suppression of REM during waking is probably mediated in part via excitation of acetylcholine-inhibited GABAergic neurons.

The basal ganglia are involved not only with motor processes such as posture, pre-movement planning and movement initiation, but also with the processing and modulation of nociceptive somatosensory information. In the current studies, unilateral, intrastriatal 6-hydroxydopamine (6-OHDA) was used to investigate how dopamine depletion alters nociceptive behavioral responses to chemical, thermal and mechanical stimulation in rats. Compared to control rats injected with intrastriatal saline, rats depleted of dopamine displayed increased nociceptive responses to chemical stimulation of the face and hyperalgesic responses to thermal stimulation of the hind paw without alterations in rearing behavior or body weight gain. Minor changes were observed in the response to mechanical stimulation of the hind paws and face. These data provide further evidence that the dopaminergic nigrostriatal pathway plays a role in the modulation of nociceptive information.

Estradiol valerate (EV)-induced polycystic ovaries (PCO) in rats are associated with higher ovarian release and content of norepinephrine, decreased beta2-adrenoceptors (ARs), and dysregulated expression of alpha1-AR subtypes, all preceded by an increase in the production of ovarian NGF. The aim of this study was to further elucidate the role of NGF in the ovaries by blocking the action of NGF during development of EV-induced PCO in rats. Control and EV-injected rats were treated with intraperitoneal injections of IgG (control and PCO groups) or with anti-NGF antibodies (anti-NGF and PCO anti-NGF groups) every third day for 5 wk starting from the day of PCO induction. Rat weight, estrous cyclicity, ovarian morphology, ovarian mRNA, and protein expression of alpha1-AR subtypes, beta2-AR, the NGF receptor tyrosine kinase A (TrkA), p75 neurotrophin receptor (p75NTR), and tyrosine hydroxylase (TH) were analyzed. Ovaries in both PCO and PCO anti-NGF groups decreased in size as well as in number and size of corpora lutea. mRNA expression of alpha1a-AR and TrkA in the ovaries was lower, whereas expression of alpha1b- and alpha1d-AR and TH was higher, in the PCO group than in controls. Protein quantities of alpha1-ARs, TrkA, p75NTR, and TH were higher in the PCO group compared with controls, whereas the protein content of beta2-AR was lower. Anti-NGF treatment in the PCO group restored all changes in mRNA and protein content, except that of alpha1b-AR and TrkA mRNAs, to control levels. The results indicate that the NGF/NGF receptor system plays a role in the pathogenesis of EV-induced PCO in rats.

Polycystic ovary syndrome (PCOS) is the main cause of infertility in women. Despite extensive research aimed at identifying the pathogenetic mechanism underlying this condition, the aetiology of the disease is still unknown. Evidence from studies on women with PCOS and on an experimental rat polycystic ovary (PCO) model suggests that the sympathetic regulatory drive to the ovary may be unbalanced. The present study was designed to investigate this hypothesis. Accordingly, we used the well-defined rat PCO model, where PCO is induced by a single intramuscular (i.m.) injection of estradiol valerate (EV), and compared the model with oil-injected controls. We studied the ovarian expression of the alpha1- and beta2-adrenoceptors (ARs), the neurotrophin receptor p75 (p75NTR), and the sympathetic marker tyrosine hydroxylase (TH) at two time points: 30 and 60 days after EV injection. Our data demonstrate for the first time that all of the alpha1-AR subtypes are expressed in normal rat ovaries at both the mRNA and the protein levels. Furthermore, the expression of the alpha1-AR subtypes was differentially modulated in a time- and subtype-dependent manner in rats with EV-induced PCO. The ovaries in rats with steroid-induced PCO are characterised by an early overexpression of these molecules and p75NTR, while the beta2-AR was downregulated. An increase in the expression of ovarian TH after EV injection was also detected, suggesting a structural and functional remodelling of ovarian sympathetic innervation in PCO rats. Our evidence strongly indicates that the role of the sympathetic nervous system is crucial in the pathogenesis of EV-induced PCO. Overall, our findings suggest that therapeutical approaches aimed at down-regulating the sympathetic tone to the ovary could be useful in the prevention and clinical treatment of PCOS.

A synthetic peptide corresponding to residues 32-47 of rat tyrosine hydroxylase (TH) was phosphorylated by protein kinase A at Ser40 and used to generate antibodies in rabbits. Reactivity of the anti-pTH32-47 antibodies with phospho- and dephospho-Ser40 forms of TH protein and peptide TH32-47 was compared with reactivity of antibodies to nonphosphorylated peptide and to native TH protein. In antibody-capture ELISAs, anti-pTH32-47 was more reactive with the phospho-TH than with the dephospho-TH forms. Conversely, antibodies against the nonphosphorylated peptide reacted preferentially with the dephospho-TH forms. In western blots, labeling of the approximately 60-kDa TH band by anti-pTH32-47 was readily detectable in lanes containing protein kinase A-phosphorylated native TH at 10-100 ng/lane. In blots of supernatants prepared from striatal synaptosomes, addition of a phosphatase inhibitor was necessary to discern labeling of the TH band with anti-pTH32-47. Similarly, anti-pTH32-47 failed to immunoprecipitate TH activity from supernatants prepared from untreated tissues, whereas prior treatment with either 8-bromoadenosine 3',5'-cyclic monophosphate or forskolin enabled removal of TH activity by anti-pTH32-47. Lastly, in immunohistochemical studies, anti-pTH32-47 selectively labeled catecholaminergic cells in tissue sections from perfusion-fixed rat brain.

A peptide corresponding to position 32-47 in tyrosine hydroxylase was synthesized (TH-16) and polyclonal antibodies against this peptide were raised in rabbits (anti-TH-16). The effects of anti-TH-16 on modulation of tyrosine hydroxylase activity were investigated. Anti-TH-16 enhanced the enzymatic activity in a concentration-dependent manner, and the antigen TH-16 inhibited the stimulatory activity of the antiserum in a concentration-dependent manner. The activated enzyme had a lower Km app for the cofactor 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropterin and a higher Vmax app than the nonactivated enzyme. Anti-TH-16 was characterized further by its ability to immunoprecipitate the enzyme activity by labeling tyrosine hydroxylase after Western blotting and by immunohistochemical labeling of catecholaminergic neurons. Anti-TH-16 did not block activation of tyrosine hydroxylase by phosphorylation catalyzed by cyclic AMP-dependent protein kinase. Exposure of the enzyme to anti-TH-16 and subsequent phosphorylation of the enzyme resulted in a greater activation of the enzyme than the sum of activation produced by these two treatments separately. However, the activation was less than additive when the enzyme was first phosphorylated and subsequently exposed to anti-TH-16. The present study demonstrates the utility of anti-TH-16 in investigating the molecular aspects of the enzyme activation.