Airway responses to capsaicin in guinea pigs: role of NK-1 and NK-2 neurokinin receptors

The role of NK-1 and NK-2 receptors on the pulmonary response to capsaicin in guinea pigs was evaluated using intravenous infusion of selective nonpeptide antagonists of NK 1 (CP 96345, 300 nmol/kg, and SR 140333, 300 nmol/kg) and NK-2 (SR 48968, 100 nmol/kg) neurokinin receptors. Maximal values of pulmonary dynamic elastance (Edyn) and pulmonary resistance (RL) after capsaicin infusion were significantly lower in the presence of SR 48968 (p < .005). Morphometric analysis of lungs obtained by quick-freezing showed significant attenuation of airway contraction and peribronchiolar edema formation in the presence of NK-2 antagonist (p < .001). When compared to guinea pigs that received only capsaicin, animals that received SR 140333 or CP 96345 showed lower values of Edyn, RL, airway contraction, and peribronchiolar edema, but only the difference in Edyn values was significant. The combination of NK-1 and NK-2 antagonists was not more effective than NK-2 antagonist alone in attenuating capsaicin effects. The results suggest that airway effects of capsaicin are mainly mediated by activation of NK-2 receptors although NK-1 receptors may also play a role.     

Inhibition of tuberoinfundibular dopaminergic neural activity during suckling: involvement of mu and kappa opiate receptor subtypes

Previous studies have shown that mu (mu) and kappa (kappa) opioid antagonists inhibit suckling-induced prolactin release. Prolactin responses elicited by pup suckling or opioid administration are mediated, at least in part, by suppression of dopamine (DA) release from tuberoinfundibular dopaminergic (TIDA) neurons in the hypothalamus. We examined the effects of the mu opiate receptor antagonist, beta-funaltrexamine (beta-FNA), and the kappa opiate receptor antagonist, nor-binaltorphimine (nor-BNI) on the activity of TIDA neurons in lactating rats. TIDA neuronal activity was determined by measuring DOPA accumulation in the caudate putamen (CP) and median eminence (ME). The effects of opioid antagonist treatment were determined in pup-deprived (low circulating prolactin levels) or pup-suckled rats (high circulating prolactin levels). The accumulation of 5-hydroxytryptophan (5-HTP) in the medial preoptic area (MPOA), the anterior hypothalamus (AH) and the median eminence (ME) was quantified as an index of serotonergic activity in the same animals for comparative purposes. In vehicle treated rats, suckling caused a significant and selective decrease in DOPA accumulation in the ME. beta-FNA (5 micrograms, i.c.v.) pretreatment significantly increased DOPA accumulation in the ME of pup-deprived and pup-suckled rats. beta-FNA pretreatment also prevented the suckling-induced suppression of DOPA accumulation in the ME. In contrast to the actions of beta-FNA, pretreatment with nor-BNI (8 micrograms, i.c.v.) did not significantly affect the activity of the TIDA neurons in pup-deprived or pup-suckled rats. Suckling alone did not alter 5-HTP accumulation in any of the brain regions examined, and neither opioid antagonist had appreciable effects on 5-HTP accumulation. These results demonstrate that the EOP tonically inhibit the TIDA neurons in both pup-deprived and pup-suckled, post-partum female rats by acting through the mu, but not the kappa, opiate receptor subtype. Furthermore, the suckling-induced inhibition of TIDA neurons is also mediated through the EOP acting at mu, but not kappa opioid receptors.     

Serotonin receptors: subtypes, functional responses and therapeutic relevance

Recent, rapid progress in the molecular biology of serotonin (5-HT) receptors requires conceptual re-thinking with respect to receptor classification. Thus, based on operational criteria (agonist and antagonist rank order), as well as transduction mechanisms involved and the structure of the receptor protein, the Nomenclature Committee of the Serotonin Club has proposed the following classification and nomenclature: the main receptor types 5-HT1 to 5-HT4, recombinant receptors (e.g. 5-ht5 to 5-ht7) and 'orphan' receptors. The aim of the present review is to discuss the events leading to this classification, the criteria for and functional responses mediated by various 5-HT receptors, as well as the therapeutic possibilities with 5-HT ligands.     

Switching of chemoattractant receptors programs development and morphogenesis in Dictyostelium: receptor subtypes activate common responses at different agonist concentrations

Although uranium (U) is a classic experimental nephrotoxin, there are few data on its potential long-term chemical toxicity. These studies were undertaken to derive a no-observed-adverse-effect level (NOAEL) in male and female Sprague-Dawley rats following 91-day exposure to uranium (as uranyl nitrate hexahydrate, UN) in drinking water. Following a 28-day range-finding study, five groups of 15 male and 15 female weanling rats were exposed for 91 days to UN in drinking water (0.96, 4.8, 24, 120, or 600 mg UN/L). A control group was given tap water (< 0.001 mg U/L). Daily clinical observations were recorded. Following the study, animals were euthanized and exsanguinated, and multiple hematological and biochemical parameters were determined. Necropsies were conducted, and multiple tissues were sampled for histopathological examination. The hematological and biochemical parameters were not affected in a significant exposure-related manner. Although there were qualitative and slight quantitative differences between males and females, histopathological lesions were observed in the kidney and liver, in both males and females, in all groups including the lowest exposure groups. Renal lesions of tubules (apical nuclear displacement and vesiculation, cytoplasmic vacuolation, and dilation), glomeruli (capsular sclerosis), and interstitium (reticulin sclerosis and lymphoid cuffing) were observed in the lowest exposure groups. A NOAEL was not achieved in this study, since adverse renal lesions were seen in the lowest exposed groups. A lowest-observed-adverse-effect level of 0.96 mg UN/L drinking water can be reported for both the male and the female rats (average dose equivalent 0.06 and 0.09 mg U/kg body wt/day, respectively).     

Discriminative stimulus properties of (±)-fenfluramine: The role of 5-HT? receptor subtypes.

The role of serotonin 5-HT? receptors (5-HT?R) in the discriminative stimulus effects of fenfluramine was investigated. Male Sprague-Dawley rats were trained to discriminate (±)-fenfluramine (2 mg/kg ip) from saline using a 2-lever, water-reinforced paradigm. Drug-lever responding after fenfluramine was dose-dependent. The 5-HT2C/1BR agonist mCPP and the 5-HT2CR agonist MK 212 fully substituted, whereas the 5-HT2A/2CR agonist DOI partially substituted, for the training drug. The 5-HT2BR agonist BW 723C86 engendered saline-lever responding. The 5-HT2C/2BR antagonist SB 206553 completely antagonized the fenfluramine discrimination as well as the full substitutions of mCPP and MK 212 and the partial substitution of DOI. The selective 5-HT2AR antagonist M100907 partially suppressed the stimulus effects of fenfluramine, mCPP, and MK 212 and almost fully attenuated the partial substitution of DOI. RS 102221, a selective 5-HT2CR antagonist that does not cross the blood-brain barrier, did not alter the fenfluramine cue. Results demonstrate that the discriminative stimulus effects of fenfluramine are centrally mediated by 5-HT2CR and to some extent by 5-HT2AR. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The kappa-opioid receptor: evidence for the different subtypes

Classification of drugs acting on the kappa-opioid receptors seems to be difficult, since some of these ligands are also sigma agonists and/or display non-opioid actions as well. Furthermore, certain benzomorphans having kappa-agonistic character, are shown to be mu-antagonists too. Therefore the classification of the kappa-opioid receptor has to be presently restricted to two subclasses that also have physiological meaning. Dynorphin and Met-enkephalin-Arg6-Phe7 are proposed as endogenous peptide ligands for kappa-receptors. Nonpeptide agonists are benzeneacetamides interacting with the kappa1 receptor. Benzomorphans bind to both subtypes of kappa-receptors. No selective nonpeptide ligand for the kappa2 receptor exists as yet. Nor-binaltorphimine, a specific kappa-antagonist also inhibits both kappa-subtypes. Further research for kappa2 selective drugs is necessary for clear distinction between the two kappa-opioid binding sites. Molecular cloning of opioid receptors including their subtypes are expected to provide direct proof of their existence.     

Naturally occurring analogs of lysophosphatidic acid elicit different cellular responses through selective activation of multiple receptor subtypes

Lysophosphatidic acid (LPA), plasmalogen-glycerophosphate (alkenyl-GP) and, cyclic-phosphatidic acid (cyclic-PA) are naturally occurring phospholipid growth factors (PLGFs). PLGFs elicit diverse biological effects via the activation of G protein-coupled receptors in a variety of cell types. In NIH3T3 fibroblasts, LPA and alkenyl-GP both induced proliferation, whereas cyclic-PA was antiproliferative. LPA and alkenyl-GP decreased cAMP in a pertussis toxin-sensitive manner, whereas cyclic-PA caused cAMP to increase. LPA and alkenyl-GP both stimulated the activity of the mitogen-actived protein kinases extracellular signal regulated kinases 1 and 2 and c-Jun NH2-terminal kinase, whereas cyclic-PA did not. All three PLGFs induced the formation of stress fibers in NIH3T3 fibroblasts. To determine whether these lipids activated the same or different receptors, heterologous desensitization patterns were established among the three PLGFs by monitoring changes in intracellular Ca2+ in NIH3T3 fibroblasts. LPA cross-desensitized both the alkenyl-GP and cyclic-PA responses. Alkenyl-GP cross-desensitized the cyclic-PA response, but only partially desensitized the LPA response. Cyclic-PA only partially desensitized both the alkenyl-GP and LPA responses. We propose that pharmacologically distinct subsets of PLGF receptors exist that distinguish between cyclic-PA and alkenyl-GP, but are all activated by LPA. We provide evidence that the PSP24 receptor is selective for LPA and not activated by the other two PLGFs. RT-PCR and Northern blot analysis indicate the co-expression of mRNAs encoding the EDG-2, EDG-4, and PSP24 receptors in a variety of cell lines and tissues. However, the lack of mRNA expression for these three receptors in the LPA-responsive Rat-1 and Sp2-O-Ag14 cells suggests that a number of PLGF receptor subtypes remain unidentified.     

Airway responses in healthy and asthmatic subjects following exposure to low ambient air temperature

Agonist-activated Ca2+ entry is important in many biological responses such as secretion and cell growth(1,2). In nonexcitable cells which have no voltage-operated Ca2+ channels (VOCC), agonist-receptor interaction can trigger Ca2+ entry across the plasmalemma via several entry pathways(1-3) (Fig 1): (A) channels which are intrinsic structures of the receptor (receptor-operated channels), (B) channels which are coupled to receptors via a G-protein (G-protein-operated channels), (C) channels which are activated by some second messengers (second-messenger-operated channels), and (D) channels which open upon intracellular nonmitochondrial Ca2+ store depletion (Ca2+ release-activated channels) resulting from inositol 1, 4, 5-trisphosphate-induced Ca2+ release or inhibition of Ca2+ re-uptake (see next section). Ca2+ entry via the 4th type of channel, also known as "capacitative Ca2+ entry" (CCE)[4], has aroused much interest in the past decade because of its intriguing nature as retrograde signalling. In this brief review, we present the evidence for and the possible biochemical processes involved in CCE. We also discuss the use of 2 novel Ca2+ entry blockers: tetrandrine and SK&F 96365. Emphasis will be put on the human leukemic HL-60 cell line, a popular cell system for intracellular Ca2+ homeostasis studies and also a model the signal transduction of which we have been investigating during the past few years.     

Amino acids of the alpha1B-adrenergic receptor involved in agonist binding: differences in docking catecholamines to receptor subtypes

Site-directed mutagenesis and molecular dynamics analysis of the 3-D model of the alpha1B-adrenergic receptor (AR) were combined to identify the molecular determinants of the receptor involved in catecholamine binding. Our results indicate that the three conserved serines in the fifth transmembrane domain (TMD) of the alpha1B-AR play a distinct role in catecholamine binding versus receptor activation. In addition to the amino acids D125 in TMDIII and S207 in TMDV directly involved in ligand binding, our findings identify a large number of polar residues playing an important role in the activation process of the alpha1B-AR thus providing new insights into the structure/function relationship of G protein-coupled receptors.     

Cognitive and behavioral responses to illness information: the role of health anxiety

The cognitive-behavioral theory of health anxiety predicts atypical responses in health anxious individuals when exposed to health related information. Systematic research is still needed to support the theory. This investigation examined 192 participants varying a subclinical levels of health anxiety, who were randomly given feedback on an ostensible diagnostic measure, indicating positive, negative or ambiguous risk for health complications. Responses to a cold pressor task were then measured. The results indicated that regardless of the type of feedback patients were given, health anxious individuals displayed the predicted cognitive (e.g. negatively interpreted information) and behavioral responses (e.g. increased reassurance seeking). Important and perhaps central cognitions to health anxiety were identified. Health anxious individuals regarded themselves to be at greater risk for disease overall, and attached greater accuracy to health related information. Extending the cognitive-behavioural theory, health anxiety was found to be associated with decreased usage of positive somatic monitoring of symptoms, suggesting health anxiety may be associated with a failure to engage in protective strategies. Health anxiety did not result in cognitive or behavioural avoidance of illness information. Clinical implications and future directions for research are described.     

Prostaglandin E receptor subtypes in cultured rat microglia and their role in reducing lipopolysaccharide-induced interleukin-1beta production

Prostaglandins (PGs) are potent modulators of brain function under normal and pathological conditions. The diverse effects of PGs are due to the various actions of specific receptor subtypes for these prostanoids. Recent work has shown that PGE2, while generally considered a proinflammatory molecule, reduces microglial activation and thus has an antiinflammatory effect on these cells. To gain further insight to the mechanisms by which PGE2 influences the activation of microglia, we investigated PGE receptor subtype, i.e., EP1, EP2, EP3, and EP4, expression and function in cultured rat microglia. RT-PCR showed the presence of the EP1 and EP2 but not EP3 and EP4 receptor subtypes. Sequencing confirmed their identity with previously published receptor subtypes. PGE2 and the EP1 agonist 17-phenyl trinor PGE2 but not the EP3 agonist sulprostone elicited reversible intracellular [Ca2+] increases in microglia as measured by fura-2. PGE2 and the EP2/EP4-specific agonists 11-deoxy-PGE1 and 19-hydroxy-PGE2 but not the EP4-selective agonist 1-hydroxy-PGE1 induced dose-dependent production of cyclic AMP (cAMP). Interleukin (IL)-1beta production, a marker of activated microglia, was also measured following lipopolysaccharide exposure in the presence or absence of the receptor subtype agonists. PGE2 and the EP2 agonists reduced IL-1beta production. IL-1beta production was unchanged by EP1, EP3, and EP4 agonists. The adenylyl cyclase activator forskolin and the cAMP analogue dibutyryl cAMP also reduced IL-1beta production. Thus, the inhibitory effects of PGE2 on microglia are mediated by the EP2 receptor subtype, and the signaling mechanism of this effect is likely via cAMP. These results show that the effects of PGE2 on microglia are receptor subtype-specific. Furthermore, they suggest that specific and selective manipulation of the effects of PGs on microglia and, as a result, brain function may be possible.     

G-protein-coupled receptor regulation: role of G-protein-coupled receptor kinases and arrestins

G-protein-coupled receptors (GPCRs) represent a large family of proteins that transduce extracellular signals to the interior of cells. Signalling through these receptors rapidly desensitized primarily as the consequence of receptor phosphorylation, but receptor sequestration and downregulation can also contribute to this process. Two families of serine/threonine kinases, second messenger dependent protein kinases and receptor-specific G-protein-coupled receptor kinases (GRKs), phosphorylate GPCRs and thereby contribute to receptor desensitization. Receptor-specific phosphorylation of GPCRs promotes the binding of cytosolic proteins referred to as arrestins, which function to further uncouple GPCRs from their heterotrimeric G-proteins. To date, the GRK protein family consists of six members, which can be further classified into subgroups according to sequence homology and functional similarities. The arrestin protein family also comprises six members, which are subgrouped on the basis of sequence homology and tissue distribution. While the molecular mechanisms contributing to GPCR desensitization are fairly well characterized, little is known about the mechanism(s) by which GPCR responsiveness is reestablished, other than that receptor sequestration (internalization) might be involved. The goal of the present review is to overview current understanding of the regulation of GPCR responsiveness. In particular, we will review new evidence suggesting a pleiotropic role for GRKs and arrestins in the regulation of GPCR responsiveness. GRK-mediated phosphorylation and arrestin binding are not only involved in the functional uncoupling of GPCRs but they are also intimately involved in promoting GPCR sequestration and as such likely play an important role in mediating the subsequent resensitization of GPCRs.     

Cellular responses to nitric oxide: role of protein S-thiolation/dethiolation

Nitrosothiols, the product of the reaction of nitric oxide-derived species (NOx) with thiols, participate in both cell signaling and cytotoxic events. Glutathione has recently been shown to modulate nitrosothiol-mediated signal transduction and to protect against NOx-mediated cytotoxicity. We have investigated the role of protein S-thiolation/dethiolation as a potential mechanism by which glutathione regulates nitrosothiol signaling and toxicity. Our data show that exogenous sources of NOx decreased both free protein thiol and total glutathione levels in endothelial cells. The decrease in glutathione levels could not be accounted for by formation of S-nitrosoglutathione (GSNO) since borohydride treatment of the nonprotein fraction of cell extracts did not restore glutathione levels, whereas borohydride treatment of protein-containing cell extracts led to recovery of glutathione levels. The NOx-mediated decrease in glutathione and protein thiol content was correlated with an increase in protein mixed disulfide formation, as measured by the incorporation of [35S]glutathione into cellular proteins. [35S]glutathione was incorporated into proteins via a covalent disulfide bond since dithiothreitol removed the radiolabel from cellular proteins. The withdrawal of the exogenous NOx source led to recovery of free protein thiol and cellular glutathione levels, which correlated with the dethiolation of proteins. Dethiolation required the action of the glutathione redox system since 1, 3-bis(2-chloroethyl)-1-nitrosourea, an inhibitor of glutathione reductase, blocked both the recovery of glutathione levels and the dethiolation of proteins. These results suggest that exposure of cells to NOx does not lead to accumulation of GSNO but rather stimulates protein S-thiolation, a mechanism which may have important implications with respect to nitrosothiol signaling and toxicity.     

Light microscope autoradiography of peripheral dopamine receptor subtypes

Radioligand binding assay techniques associated with light microscope autoradiography were used for investigating the pharmacological profile and the micro anatomical localization of peripheral dopamine receptor subtypes. In systemic arteries, the predominant dopamine D1-like receptor belongs to the D5 (or D1B) subtype. It is located within smooth muscle of the tunica media. In pulmonary arteries, dopamine D1-like receptors have primarily an endothelial localization and belong to the dopamine D1 (or D1A) receptor subtype. Both systemic and pulmonary arteries express a dopamine D2-like receptor belonging to the D2 receptor subtype. It has a prejunctional localization in the majority of vascular beds investigated. In cerebral, coronary and mesenteric arteries, it has also an endothelial localization. In the heart, a dopamine D4 receptor was identified. It is expressed by atrial tissue and has a widespread distribution overall atrial musculature. The kidney expresses both dopamine D1-like and D2-like receptors. Renal dopamine D1-like receptors have a vascular and tubular localization. The majority of these sites belongs to the D5 receptor subtype. A smaller D1 receptor population has primarily a tubular localization. Renal dopamine D2-like receptors belong to the dopamine D3 subtype and in lesser amounts to the D2 and D4 receptor subtypes. Renal dopamine D3 receptor has to a greater extent a tubular localization, whereas the D4 receptor is located within glomerular arterioles. The above results suggest that radioligand binding assay and autoradiographic techniques, if performed in the presence of compounds displaying specific receptor subtype selectivity, may contribute to characterize, mainly from a quantitative point of view, peripheral dopamine receptors.     

Postnatal development of delta-opioid receptor subtypes in mice

1. The density and affinity of binding sites for the delta-selective opioid ligands [3H]-[D-Ala2, Asp4]deltorphin (DELT-I), [3H]-[D-Ala2Glu4]-deltorphin (DELT-II), [3H]-[D-Pen2,D-Pen5]enkephalin (DPDPE), and [3H]-naltrindole (NTI) were determined in whole brain from 10, 15, 25 and 60 day-old C57BL mice. 2. At all ages, the analyses of the homologous displacement curves, gave best fits to single rather than to multiple site models. The binding capacity (Bmax) labelled by [3H]-NTI was about one half that labelled by [3H]-DELT-I, [3H]-DELT-II and [3H]-DPDPE. In 25 and 60 day-old mouse brain the DPDPE Bmax was 25% less than the deltorphin-II Bmax. 3. In saturation experiments, specific binding of [3H]-DELT-I on adult mouse brain homogenates was best fitted by a two-site model (34%, high affinity site, Kd = 1.08 nM and 66% low affinity sites, Kd = 39.9 nM). 4. DPDPE produced a biphasic inhibition of specific [3H]-DELTI-I binding, from 15 days of age onwards. The relative percentage of high and low affinity sites was 72% and 28% in 15 day-, 65% and 35% in 25 day- and 30% and 70% in 60 day-old mice. 5. In adult mouse brain labelled with [3H]-DELT-I, DELT-II recognized 71% of high-affinity and 29% of low-affinity sites DELT-I and DPDPE produced monophasic inhibition of specific [3H]-DELT-II binding to brain homogenates of adult mice. 6. These data suggest that a sub-population of delta-sites (probably the delta 2-subtype), recognized by DELT-I, with high affinity for DELT-II and low affinity for DPDPE develops from 25 days onward. 7. In electrically stimulated mouse vas deferens (MVD) the rank order of potency of the three delta-agonists was: DELT-I > DELT-II > DPDPE in 10 day-old mice: and DELT-I- DELT-II > DPDPE, from 25 days onward. During this time, the potency of DELT-II increased about 15 fold whereas the potency of DELT-I and DPDPE increased only 5 times. The higher efficacy of DELT-II could depend on receptor maturation towards the delta 2-subtype.