Localization of galanin and its binding sites in the quail brain

Avian galanin was first isolated from the chicken intestine, and subsequently we isolated the same peptide from the quail oviduct. Galanin is known to be a multifunctional peptide in the neuroendocrine system in mammals, while little information is available for the galanin action in the central nervous system in birds. In the present study, therefore, we examined the presence and the localization of galanin and its binding sites in the quail brain. The binding of [¹²⁵I]iodo-avian galanin to brain tissues specifically inhibited as a function of the concentrations of unlabeled avian galanin. In all of the observed brain regions, the number of galanin-binding sites when compared on the basis of unit weight was maximal in the interbrain including the preoptic and hypothalamic regions and minimal in the cerebellum. The Scatchard plot analysis of the galanin binding to the interbrain revealed that the apparent equilibrium constant of dissociation (K_d was 0.281 (95% confidence interval, 0.234-0.351) nM, suggesting the presence of a single class of high-affinity binding sites. Although galanin-like immunoreactivity was found in several restricted regions throughout the brain, the most intense immunoreaction was present in the diencephalic region. Abundant immunoreactive cell bodies and/or fibers were localized in several preoptic and hypothalamic nuclei, i.e., the nucleus preopticus medialis (PONI), the nucleus paraventricularis (PVN), the nucleus periventricularis hypothalami (PHN) and the nucleus infundibuli (IN), that are known to be involved in the control of reproductive behaviors as well as the pituitary hormone secretion. A substantial number of immunoreactive cell bodies and/or fibers was further observed within the septal nuclei in the telencephalon and several mesencephalic nuclei including the nucleus intercollicularis (ICo) involved in the control of vocalizations. These results suggest that avian galanin may act as an important factor to modulate the some reproductive behaviors and/or the pituitary hormone secretion in the bird.     

甘丙肽拮抗抑郁症的研究进展

脑内给予甘丙肽(GAL)能够调节啮齿类模型动物的抑郁应激反应,表明甘丙肽具有调节抑郁行为的作用.GAL具有GALR1、GALR2、GALR3三种亚型受体,GALR2特异性激动剂和GALR1/GALR3特异性拮抗剂均具有很好的抗抑郁作用,表明甘丙肽系统具有治疗抑郁症的前景.     

A study of the kinetics of the interaction of spiperone with binding sites on human mononuclear cells: existence of a heterogeneous population of spiperone binding sites

The kinetics and equilibrium of the interaction of [3H]spiperone with binding sites on human mononuclear cells were studied using a competitive displacement of spiperone by ketanserin, sulpiride, haloperidol, (+)- and (-)-butaclamol. [3H]Spiperone binding sites on human mononuclear cells were shown to be not of the D2, but most probably of the 5-HT2 type. The process of [3H]spiperone binding to these sites conforms to the model of ligand interaction with two independent binding sites (the high-affinity site, Kd1 = 3 nM and the low-affinity site, Kd2 = 20 nM), thus suggesting heterogeneity of spiperone sites on mononuclear cells. We found that in contrast to high-affinity binding sites, low-affinity sites show an essentially lower stability during storage of mononuclear cells.     

Galanin inhibits the potassium-evoked release of acetylcholine and the muscarinic receptor-mediated stimulation of phosphoinositide turnover in slices of monkey hippocampus

In the ventral hippocampus of Cynomologus monkey, galanin, a 29 amino acid long neuropeptide, reduced the potassium-evoked release of [3H]acetylcholine from slices preloaded with [3H]choline and diminished the carbachol-stimulated accumulation of [3H]inositol polyphosphates in hippocampal microprisms preincubated with myo-[2-3H]inositol. Using receptor autoradiography a strong, specific binding of iodinated galanin was observed in the molecular layer of the dentate gyrus. These may thus be the sites where galanin exerts its inhibitory effects on acetylcholine (ACh) release and phosphoinositide breakdown. These data provide evidence that galanin is a modulator of cholinergic function in septo-hippocampal neurons of primates.     

Involvement of the lysine-binding sites of plasminogen on its interaction with concanavalin A

Two different interactions are involved in the binding of plasminogen to concanavalin A-Sepharose: both variants 1 and 2 interact with the lectin through the lysine-binding sites and, in addition, variant 1 binds to concanavalin A due to carbohydrate recognition. Both kinds of interactions were also observed in solution by analytical ultracentrifugation. The binding of Lys-plasminogen to concanavalin A via lysine-binding sites largely exceeds that of Gluplasminogen, in accordance with the higher affinity for lysine of Lys-plasminogen. This fact can be applied to the separation of both forms of plasminogen in a single chromatographic step.     

The effects of galanin on dorsal root ganglion neurons with high glucose treatment in vitro

The exposure of neurons to high glucose concentrations is considered a determinant of diabetic neuropathy. The extracellular high concentration of glucose can cause neuronal cellular damage. Galanin (Gal) not only plays a role in processing of sensory information but also participates in energy homeostasis and glucoregulation. However, the effects of Gal on dorsal root ganglion (DRG) neurons with high glucose are not clear. Using an in vitro model of high glucose-treated DRG neurons in culture, the effects of Gal on intracellular reactive oxygen species (ROS) expression, cell viability, apoptosis, expression of Gal and its receptors (GalR1 and GalR2) of DRG neurons were investigated. Neurons were dissociated from embryonic day 15 (E15) rat DRG and cultured for 48 h and then maintained in serum-free neurobasal medium containing high glucose (45 mmol/L) or normal glucose (25 mmol/L) for 24 h. Mannitol (20 mmol/L) was also used to create a high osmotic pressure mimicking the high glucose condition. The results showed that high glucose caused a rapid increasing of intracellular ROS, decreases of cell viability, and upregulation of Gal and its mRNA. Exogenous Gal (1 μmol/L) inhibited the above effects caused by high glucose. Interestingly, high glucose caused downregulation of GalR1 and its mRNA and administration of exogenous Gal could further decrease their expression, whereas expression of GalR2 and its mRNA was not affected at different experimental conditions. The results of the present study indicate for the first time that Gal and its receptor system are involved in high glucose-induced DRG neuronal injury. The contribution of exogenous Gal on neuroprotection appears to be quite significant. These results provide rationale and experimental evidence for development and further studies of Gal on therapeutic strategy for improving diabetic neuropathy.     

Evidence for Multiple Forms of Melatonin Receptor-G-Protein Complexes by Solubilization and Gel Electrophoresis

The daily production of melatonin from the pineal gland influences circadian and seasonal behaviour and physiology. To further understand how melatonin may function, it is important to characterize the receptor and signal transduction systems. Using the detergent digitonin, we were able to solubilize the receptor from the ovine pars tuberalis (PT) membrane. The receptor was isolated as a complex associated with its heterotrimeric G-protein. In the solubilized state, pre-bound ¹²⁵l-2-iodomelatonin was stable at 4°C, but was displaceable by GTPγS. The receptor-G-protein complex could be separated by molecular mass using native polyacrylamide gel electrophoresis. We demonstrate that the receptor-complex has a molecular mass of 525 kDa and differs from solubilized receptor-complexes isolated from either the lizard brain, chicken brain or the ovine hippocampus. Furthermore the receptor complex isolated from the hippocampus had the lowest molecular mass of these tissues (365 kDa) and was found not to be sensitive to GTPγS. This may indicate the existence of a distinct non-G-protein coupled form of the receptor.     

Galanin peptide family regulation of glucose metabolism

Recent preclinical and clinical studies have indicated that the galanin peptide family may regulate glucose metabolism and alleviate insulin resistance, which diminishes the probability of type 2 diabetes mellitus. The galanin was discovered in 1983 as a gut-derived peptide hormone. Subsequently, galanin peptide family was found to exert a series of metabolic effects, including the regulation of gut motility, body weight and glucose metabolism. The galanin peptide family in modulating glucose metabolism received recently increasing recognition because pharmacological activiation of galanin signaling might be of therapeutic value to improve insuin resistance and type 2 diabetes mellitus. To date, however, few papers have summarized the role of the galanin peptide family in modulating glucose metabolism and insulin resistance. In this review we summarize the metabolic effect of galanin peptide family and highlight its glucoregulatory action and discuss the pharmacological value of galanin pathway activiation for the treatment of glucose intolerance and type 2 diabetes mellitus.     

Dynamic regulation of GABAA receptors at synaptic sites

γ-Aminobutyric acid type A receptors (GABA_ARs) mediate fast synaptic inhibition in brain and spinal cord. They are ligand-gated ion channels composed of numerous distinct subunit combinations. For efficient synaptic transmission, GABA_ARs need to be localized to and anchored at postsynaptic sites in precise apposition to presynaptic nerve terminals that release the neurotransmitter GABA. Neurons therefore require distinct mechanisms to regulate intracellular vesicular protein traffic, plasma membrane insertion, synaptic clustering and turnover of GABA_ARs. The GABA_A receptor-associated protein GABARAP interacts with the γ2 subunit of GABA_ARs and displays high homology to proteins involved in membrane fusion underlying Golgi transport and autophagic processes. The binding of GABARAP with NSF, microtubules and gephyrin together with its localization at intracellular membranes suggests a role in GABA_AR targeting and/or degradation. Growth factor tyrosine kinase receptor activation is involved in the control of GABA_AR levels at the plasma membrane. In particular insulin recruits GABA_ARs to the cell surface. Furthermore, the regulation of GABA_AR surface half-life can also be the consequence of negative modulation at the proteasome level. Plic-1, a ubiquitin-like protein binds to both the proteasome and GABA_ARs and the Plic1-GABA_AR interaction is important for the maintenance of GABA-activated current amplitudes. At synaptic sites, GABA_ARs are clustered via gephyrin-dependent and gephyrin-independent mechanisms and may subsequently become internalized via clathrin-mediated endocytosis underlying receptor recycling or degradation processes. This article discusses these recent data in the field of GABA_AR dynamics.     

Autoradiographic characterization of I-neurotensin binding sites in human entorhinal cortex

The laminar and rostro—caudal distribution of ¹²⁵I-neurotensin binding sites is described in human entorhinal cortex using quantitative autoradiography. Specific binding was most prominent over the cell clusters of layer II of the entorhinal cortex throughout its rostro-caudal extent. Dense binding was also observed in the adjacent presubiculum and cortical amygdaloid transition area, whereas minimal binding was detected in the hippocampus and dentate gyms. ¹²⁵I-Neurotensin may serve as a selective probe for neurotensin receptor alterations and layer II-specific cytoarchitectural disturbances in the entorhinal cortex in neuropsychiatric diseases associated with abnormalities of the mesial temporal lobe.     

Multiple actions of fluoride ions upon the phosphoinositide cycle in the rat brain

The effects of sodium fluoride upon basal and agonist-stimulated inositol phospholipid breakdown have been investigated in rat brain miniprisms. NaF concentration dependently increased basal inositol phospholipid breakdown, with a maximum effect being seen at 20 mM. NaF reduced the inositol phospholipid breakdown responses to stimulation by carbachol, noradrenaline, serotonin and quisqualate, but not to the stimulation produced by raising the assay [K+] from 6 to 18 mM. More detailed study demonstrated NaF to have a 'levelling' effect, reducing all InsP/(Lipid + InsP) values greater than 0.15 (i.e. produced by carbachol at raised [K+], noradrenaline and by 50 mM K+) to about this value. Time-course experiments indicated that NaF treatment reduced the rate of carbachol-stimulated inositol phospholipid breakdown up to this InsP/(Lipid + InsP) level and thereafter blocked further breakdown. Inhibitory effects upon carbachol-stimulated inositol phospholipid breakdown were not seen with forskolin, sodium nitroprusside or 8BrcGMP. Under conditions where there is no de novo synthesis of phosphoinositides from [3H]myo-inositol, NaF reduced the total Lipid + InsP labelling by about 20%. NaF in addition inhibits the activity of Ins(1,4)P2-phosphatase in cerebral cortical homogenates. It is concluded that fluoride ions inhibit agonist-stimulated inositol phospholipid breakdown via actions not only on G-proteins but also on phosphoinositide-specific phospholipase C substrate availability.     

Selective effects of long-term lithium and carbamazepine administration on G-protein subunit expression in rat brain

The efficacy of lithium and carbamazepine in treatment of bipolar affective disorder is well established. Although a number of biochemical effects have been found the exact molecular mechanisms underlying their therapeutic actions have not been elucidated. Nor have the target regions in the brain been located. The objectives of the present investigation were to identify the selective effects and target regions of long-term treatment, with either lithium or carbamazepine, on G-protein subunit expression in rat brain. Effects were measured in hippocampus, hypothalamus, amygdala, frontal cortex, neostriatum, thalamus, raphe nuclei and cerebellum. At the protein level amounts of Gα_o decreased significantly (P<0.01) in neostriatum and Gβ increased in frontal cortex in response to both drug treatments. At the mRNA level amounts of Gα_i1 increased significantly (P<0.01) in neostriatum. Gα_s messenger amounts decreased in frontal cortex and increased in thalamus. These effects were common for both drugs, however, in addition also some differential effects, specific for either of the two drugs, were observed. We conclude frontal cortex and neostriatum are important target regions of long-term treatment with either lithium or carbamazepine and suggest Gα_o, Gα_s, Gα_i1 and Gβ to be selective target molecules.     

甘丙肽系统在雌性焦虑抑郁样小鼠海马中的表达和作用

目的研究在雌性焦虑抑郁样行为小鼠海马中甘丙肽及其受体的表达和所起的作用。方法制备甘丙肽及甘丙肽受体-1(galanin receptor subtybe 1,Gal R1)、受体-2(Gal R2)、受体-3(Gal R3)探针,运用原位杂交技术观察甘丙肽及其受体Gal R1、Gal R2、Gal R3在雌性C57BL/6J小鼠海马中分布情况。另取雌性C57BL/6J小鼠随机分为实验组(15只)与对照组(15只),实验组给予慢性温和型不可预见性刺激(chronic unpredictability mild stress,CUMS)4周,对照组不给予任何刺激。4周后分别行糖水偏好实验、新物体识别实验、旷场实验及悬尾实验以评估小鼠的焦虑抑郁样行为水平及工作记忆情况;行为实验后,每组8只小鼠提取海马RNA行qPCR以检测海马甘丙肽及其受体表达情况,其余小鼠取脑行免疫荧光染色检测海马C-Fos阳性细胞数。结果原位杂交结果显示甘丙肽及其受体Gal R1、Gal R2在腹侧海马Hilus区有表达,Gal R3未见阳性信号。糖水偏好实验实验组较对照组糖水饮用量减少(P0.05);新物体识别实验显示,实验组比对照组探索新物体时间百分比短(P0.05);旷场实验中,实验组小鼠较对照组中间停留时间缩短(P0.05);悬尾实验中,实验组较对照组累计静止时间延长(P0.05)。qPCR结果示实验组小鼠海马甘丙肽及Gal R1表达较对照组增高(P0.05)。免疫组化结果示实验组小鼠海马齿状回C-Fos阳性细胞较对照组增多(P0.05)。结论甘丙肽、Gal R1及Gal R2主要分布在小鼠海马的腹侧Hillus区;在雌性C57BL/6J小鼠中,海马甘丙肽及Gal R1的增加可能与焦虑抑郁样行为有关,与海马齿状回C-Fos细胞增加也有关。     

Characterization of [3H]AF-DX 116 binding sites in the rat brain: Evidence for heterogeneity of muscarinic-M2 receptor sites

This study shows that [³H]AF-DX 116 binds specifically, saturably, and with high affinity to putative muscarinic-M2 receptor sites in the rat brain. In homogenates of the hippocampus, cerebral cortex, striatum, thalamus, and cerebellum, [³H]AF-DX 116 appears to bind two subpopulations of muscarinic sites: one class of higher affinity sites (K_d < 4.0 nM) and one class of lower affinity sites (K_d > 50 nM, except in the cerebellum). The apparent maximal capacities (B_max) of [³H]AF-DX 116 sites in forebrain tissues ranged between 34 and 69 fmol/mg protein for the higher affinity site, and between 197 and 451 fmol/mg protein for the lower affinity site. In cerebellar homogenates, the maximal capacity of [³H]AF-DX 116 binding sites was 10.4 ± 0.4 (K_d = 1.9 ± 0.2 nM) and 39.1 ± 2.6 (K_d = 26 ± 7 nM) fmol/mg protein for the higher and the lower affinity site, respectively. Determination of the K_d for the higher and lower affinity [³H]AF-DX 116 sites from association and dissociation constants yielded similar values to those obtained from the saturation data. The ligand selectivity pattern reveals that AF-DX 116 is more potent than (–)QNB > atropine > methoctramine > 4-DAMP > gallamine > NMS > carbamylcholine > oxotremorine > pirenzepine > > nicotine in competing for the higher affinity [³H]AF-DX 116 sites. With few exceptions, the pattern was similar for the lower affinity sites. For example, (–)QNB was more potent than AF-DX 116 and pirenzepine was more potent than either oxotremorine or 4-DAMP at the lower affinity [³H]AF-DX 116 sites. In addition, pirenzepine was modestly more potent at the lower compared to the higher affinity sites. Neither the higher nor the lower affinity [³H]AF-DX 116 sites were sensitive to the effects of Gpp(NH)p or N-ethylmaleimide. In addition, the carbamylcholine-induced inhibition of [³H]AF-DX 116 binding to the higher and the lower affinity sites was altered by Gpp(NH)p and NEM, to a similar extent. However, Gpp(NH)p decreased the affinity of carbamylcholine (i.e., increased the IC₅₀), whereas N-ethylmaleimide had the opposite effect. Furthermore, N-ethylmaleimide also appeared to steepen the curve for the carbamylcholine-induced inhibition of [³H]AF-DX 116 binding, as evidenced by the increased n^H. Thus it appears that [³H]AF-DX 116 binds to two subsets of muscarinic-M2 receptors in the rat brain, which can be differentiated by their affinity for certain agonists and antagonists.     

Localization and developmental pattern of vasoactive intestinal polypeptide binding sites in the human hypothalamus

Using a quantitative in vitro autoradiographic approach, vasoactive intestinal polypeptide (VIP) binding site densities were compared in the post-mortem hypothalamus of human neonate/infant and adult. The densities were similar during development in most of the hypothalamic nuclei and areas examined underlying the stability of ¹²⁵I-VIP binding sites in the post-mortem hypothalamus of young and adult individuals. However, the ventral part of the medial preoptic area, the medial, lateral, and supramammillary nuclei were characterized by an increase of ¹²⁵I-VIP binding with age. In young and adult individuals, the highest densities of hypothalamic ¹²⁵1-VIP binding sites were detected in the supraoptic and infundibular nuclei; the ependyma; the organum vasculosum of the lamina terminalis; the horizontal limb of the diagonal band of Broca; the ventral part of the medial preoptic area (in adult); the suprachiasmatic, paraventricular, and periventricular nuclei; and the medial and lateral mammillary nuclei in adult. Moderate densities were found in the vertical limb of the diagonal band of Broca, the bed nucleus of the stria terminalis, the ventral part of the medial preoptic area in neonate/infant, the medial and lateral mammillary nuclei in neonate/infant, the supramammillary nucleus in adult, the dorsal hypothalamic area, and the ventromedial nucleus. Low to moderate binding site densities were observed in the other hypothalamic regions of young or adult individuals. The nonspecific binding ranged from 15% of the total binding in the anterior hypothalamus to 20% in the mediobasal and posterior hypothalamic levels. Taken together, these results provide evidence for a large distribution of VIP binding sites in neonate/infant and adult human hypothalamus suggesting the implication of VIP in the development of this brain structure and the maintenance of its various functions. © 1994 Wiley-Liss, Inc.     

Light-microscopic localization of somatostatin binding sites in the locus coeruleus of the rat

Somatostatin (SS14) binding sites within locus coeruleus (LC) were localized at the light microscope level by [¹²⁵I][Tyr⁰,d-Trp⁸]SS14 radioautography combined with an immunohistochemical/neurotoxic lesioning approach. In intact rats, the dense accumulation of SS14 binding sites of LC conspicuously overlapped with the cluster of tyrosine hydroxylase (TH) immunoreactive neurons; SS14 specific binding was directly proportional to the number of TH immunostained (TH+) cell bodies per mg of tissue throughout LC. Complete lesion of catecholaminergic nerve cell bodies of LC by intracerebroventricular injection of 6-hydroxydopamine (6-OHDA) resulted in the total abolition of SS14 specific binding in the structure. In addition, specifically bound [¹²⁵I][Tyr⁰,d-Trp⁸]SS14 and TH+ cell density were quantified serially in a set of rats bearing various partial neurotoxic lesions; a highly significant correlation was found between the two parameters at each of the 16 coronal levels of LC examined. The coefficient of proportionality was identical at all levels. These results strongly suggest that somatostatin binding sites are uniformly localized on all noradrenergic neurons of LC.     

Multiple sites of vasopressin synthesis in the injured brain

Previous studies have indicated that the primary targets for vasopressin actions on the injured brain are the cerebrovascular endothelium and astrocytes, and that vasopressin amplifies the posttraumatic production of proinflammatory mediators. Here, the controlled cortical impact model of traumatic brain injury in rats was used to identify the sources of vasopressin in the injured brain. Injury increased vasopressin synthesis in the hypothalamus and cerebral cortex adjacent to the posttraumatic lesion. In the cortex, vasopressin was predominantly produced by activated microglia/macrophages, and, to a lesser extent, by the cerebrovascular endothelium. These data further support the pathophysiological role of vasopressin in brain injury.     

Psychophysiological effects of human-animal interaction: theoretical issues and long-term interaction effects

This paper reviews literature published on the psychophysiological effects of long-term human-animal interaction (i.e., pet ownership, pet adoption). A literature search was conducted using PsycInfo and Medline databases. Although the available evidence is far from being consistent, it can be concluded that, in some cases, long-term relationships with animals may moderate baseline physiological variables, particularly blood pressure. Results proved more coherent in studies where animals were adopted by owners as part of the procedure. This paper examines existing hypotheses seeking to account for these effects and the supporting evidence. Two major hypotheses have been suggested to explain the psychophysiological effects of long-term interaction, namely (1) stress-buffering effects of noncritical social support provided by pets; and (2) classical conditioning of relaxation. These mechanisms may partially account for the long-term health outcomes observed in a number of human-animal interaction studies.     

Oxytocin and vasopressin binding sites in the hypothalamus of the rat: Histoautoradiographic detection

Localization of oxytocin and vasopressin binding sites has so far been studied in the rat brain by means of film autoradiographs. The availability of selective iodinated ligands with high specific activity allowed us to develop the histoautoradiographical technique and to reinvestigate at the microscopic scale the distribution of these sites in the hypothalamus. Most oxytocin binding sites were localized in delimited nuclei, e.g., the medial preoptic, the ventromedial, the ventral premammillary, the supramammillary, and the medial mammillary nuclei. In addition, a weak diffuse specific labeling occurred in the medial preoptic and the anterior hypothalamic areas. The vasopressin binding sites (of the V1 a type) were detected in delimited nuclei, e.g., the suprachiasmatic, the stigmoid, and the arcuate nuclei, but they were also diffusely distributed in the lateral hypothalamic and the dorsochiasmatic areas. The locations of neurohypophysial peptides binding sites detected by light microscopy are compared with those previously obtained by film autoradiography.     

Characteristics, day-night changes, subcellular distribution and localization of melatonin binding sites in the goldfish brain

Melatonin binding sites in the goldfish brain were characterized by radioreceptor assay using 2-[¹²⁵I]iodomelatonin as the radioligand. Specific binding of 2-[¹²⁵I]iodomelatonin was rapid, stble, saturable and reversible. Saturation experiments demonstrated that 2-[¹²⁵I]iodomelatonin binds to a single class of receptor site with an affinity constant (K_rmd) of 29.8±0.7pM and a total binding capacity (B_max)of11.47 ± 0.33fmol/mg protein at mid-dark, the B_max value decreased significantly to 7.90±0.23fmol/mg protein (Pˇ0.01) with no significant variation in the K_d value (33.8±1.5pM). Competition experiments revealed the following order of pharmacological affinities: 2-iodomelatonin>melatonin> 6-hydroxymelatonin> N-acetyl-5-hydrxytryptamine> 5-methoxytryptamine> 5-methoxytryptophol> 5-methoxyindole-3-acetic acid. 5-Hydroxytryptamine, 5-hydroxytryptophol, 5-hydroxyindole-3-acetic acid, norepinephrine and acetylcholine exhibited no inhibition. Subcellular distribution of melatonin binding sites was demonstrated to be greatest in the P₂ and P₃ fractions as compared with the P₁ fraction. Localization of melatonin binding sites in discrete brain areas was determined to be highest in the optic tectum-thalamus and hypothalamus, intermediate in the telencephalon, cerebellum and medulla oblongata, and lowest in the olfactory bulbs and pituitary gland. These results suggest that characteristics of melatonin receptors are highly conserved during evolution and that in this species melatonin plays neuromodulatory roles in the central nervous system through specific receptors.