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At least 19 recordsLinked to original sources
PubMed1987

Orientation and vertical fluctuations of spin-labeled analogues of cholesterol and androstanol in phospholipid bilayers.

We have used ESR and NMR linewidth broadening by spin-labels to determine the overall orientation of spin-labeled analogues of cholesterol and androstanol in egg lecithin bilayers. While the cholesterol analogues were found to have a single orientation in each monolayer, with the acyl chain pointing towards the center of the bilayer, the androstanol analogue appeared, at least in sonicated vesicles, to experience two opposite orientations in the same monolayer, very likely with a rapid reorientation. The possibility of rapid vertical fluctuations of the sterol molecules within the phospholipid bilayer is also discussed.

Androstanols↗
PubMed2003

Complex effects of rexinoids on ligand dependent activation or inhibition of the xenobiotic receptor, CAR.

BACKGROUND: CAR/RXR heterodimers bind a variety of hormone response elements and activate transcription in the absence of added ligands. This constitutive activity of murine CAR can be inhibited by the inverse agonist ligand androstanol or increased by the agonist TCPOBOP. RXR agonists activate some RXR heterodimer complexes, which are termed permissive, while other non-permissive complexes are not responsive to such ligands. RESULTS: Direct protein-protein interaction studies demonstrate that the RXR agonist 9-cis-RA increases interaction of CAR/RXR heterodimers with the coactivator SRC-3, but also inhibits the ability of TCPOBOP to increase and androstanol to decrease coactivator binding. CAR transactivation of a response element with a five nucleotide spacer (DR-5) is unaffected by 9-cis-RA or the synthetic RXR agonist LG1069. In agreement with the inhibitory effect observed in vitro, these rexinoids block both the TCPOBOP mediated transactivation of this element and the androstanol dependent inhibition. In contrast, CAR transactivation of other response elements is increased by rexinoids. Stable expression of CAR in a HepG2 derived cell line increases expression of the endogenous CAR target CYP2B6. This expression is further increased by TCPOBOP but decreased by either androstanol or LG1069, and LG1069 blocks the stimulatory effect of TCPOBOP but not the inhibitory effect of androstanol. CONCLUSION: We conclude that CAR/RXR heterodimers are neither strictly permissive nor non-permissive for RXR signaling. Instead, rexinoids have distinct effects in different contexts. These results expand the potential regulatory mechanisms of rexinoids and suggest that such compounds may have complex and variable effects on xenobiotic responses.

Journal Article↗
PubMed2002

The role of hepatocyte RXR alpha in xenobiotic-sensing nuclear receptor-mediated pathways.

Nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) cross talk and serve as xenobiotic sensors to form a safety net against the toxic effects of harmful substances. Retinoid x receptor alpha (RXRalpha) dimerizes with CAR and PXR. In order to analyze the role of RXRalpha in these xeno-sensor-mediated pathways, hepatocyte RXRalpha-deficient mice were challenged by CAR and PXR ligands including androstanol, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), and pregnenolone 16alpha-carbonitrile (PCN). We demonstrate that hepatocyte RXRalpha deficiency prevents TCPOBOP-induced hepatomegaly and morphological changes. We also show that in vivo the cytochrome P450 (CYP) genes including CYP2A5, CYP2B10, CYP3A1, but not CYP2E1 and CYP2D6, are the RXRalpha target genes. Androstanol, TCPOBOP, and PCN can differentially regulate the expression of these CYP450 genes. In addition, the most active peroxisome proliferator activated receptor (PPARalpha) ligand, Wy14,643, also regulates some of the xeno-sensor target genes such as CYP2A5 and CYP2B10 in vivo. Thus, the ligands of different nuclear receptors can regulate common CYP450 genes and hepatocyte RXRalpha is essential for xenobiotic metabolism in vivo.

Androstanols↗
PubMed2003

The critical role of carboxy-terminal amino acids in ligand-dependent and -independent transactivation of the constitutive androstane receptor.

The mouse constitutive androstane receptor (CAR) is a unique member of the nuclear receptor superfamily, for which an inverse agonist, the testosterone metabolite 5alpha-androstan-3alpha-ol (androstanol), and an agonist, the xenobiotic 1,4-bis[2-(3, 5-dichloropyridyloxy)] benzene, are known. In this study the role of the transactivation domain 2 (AF-2) of CAR was investigated, which is formed by the seven most carboxy-terminal amino acids of the receptor. The AF-2 domain was shown to be critical for the constitutive activity by mediating a ligand-independent interaction of CAR with coactivator (CoA) proteins. In addition this domain increased and decreased contact with CoAs in the presence of agonist and inverse agonist, respectively. In analogy to classical endocrine nuclear receptors, in CAR the charge clamp between K187 (in helix 3) and E355 (within the AF-2 domain) was expected to be critical for its interaction with CoAs. However, the hydrophobic amino acids L352, L353, and I356 on the surface of the AF-2 domain were found to be more important for this protein-protein interaction. Moreover, these amino acids and C357 were shown to be involved in the response of CAR to androstanol. Interestingly, the cysteine at position 357 appears to block classical endocrine responsiveness of CAR to agonists, since mutagenesis of this amino acid both reduced CoA interaction in the absence of ligand and drastically increased inducibility by 1,4-bis[2-(3, 5-dichloropyridyloxy)] benzene. We showed that this blockade is not due to an intramolecular disulfide bridge, but is probably caused by an interaction between C357 and Y336.

Amino Acids↗
PubMed1983

An electron-spin-resonance spin-label study of the interaction of purified Mojave toxin with synaptosomal membranes from rat brain.

The structural properties of isolated purified rat brain synaptosomal membranes, both in the presence and absence of purified active toxin of the Mojave snake Crotalus scutulatus scutulatus, were studied by spin-label electron spin resonance techniques. The spectra from eight different positional isomers of nitroxide-labelled stearic acids, a rigid steroid androstanol, and a spin-labelled phosphatidylcholine intercalated into the synaptosomal membranes, were obtained as a function of temperature from 4-40 degrees C. The flexibility gradient (from spin-label order parameters) and polarity profile (from isotropic splitting factors) across the synaptosomal membranes, was characteristic for lipid bilayers. The nitroxide spin-labelled steroid, androstanol, intercalated into the synaptosomal membrane, revealed the abrupt onset of rapid cooperative rotation about the long axis of the molecule at 12 degrees C showing that the lipid molecules are rotating rapidly around their long axes at physiological temperatures. The presence of the Mojave toxin affected the synaptosomal membrane in a complex manner, depending upon the temperature and the position of the nitroxide label on the alkyl chain of the stearic acid probe. Mojave toxin exerted little effect on the flexibility gradient of the synaptosomal membrane at 20 degrees C, a temperature at which the acyl chain labels detected a structural change in the membranes. At temperatures lower than 20 degrees C, the Mojave toxin produced a change in the flexibility gradient of the synaptosomal membrane which indicated an increased disordering in the upper region of the membrane and a concomitant increased ordering of the acyl chains in the deeper regions of the membrane. At temperatures higher than 20 degrees C, the order profile of the synaptosomal membrane was shifted by the presence of the Mojave toxin in a manner which indicated that the outer parts of the membrane were more rigid and the inner regions more fluid, than in controls. A cross-over point for the perturbation occurred at C8-9, which is about 12-14 A into the membrane. This is the approximate depth of the hydrophobic pocket shown in pancreatic phospholipase A2 [Drenth et al. (1976) Nature (Lond.) 264, 373-377], a protein likely to be homologous to the basic subunit of the toxin. At all temperatures, rotational lipid motion was inhibited by the toxin as indicated by the steroid probe. The electron spin-resonance spin-label results are interpreted in terms of the partial penetration of the basic subunit of the intact toxin into the membrane, disordering the ordered chains at low temperature and ordering the disordered chains at physiological temperatures. The purified individual toxin subunits did not perturb the membrane lipids at physiological temperatures implying that both subunits must be associated for activity of the toxin which is confirmed by toxicity studies.

Animals↗
PubMed1981

Phospholipid chain immobilization and steroid rotational immobilization in acetylcholine receptor-rich membranes from Torpedo marmorata.

1. The ESR spectra of both phosphatidylcholine and phosphatidylethanolamine spin labels reveal an immobilized lipid component (tau R greater than or equal to 50 ns), in addition to a fluid component (tau R approximately 1 ns), in acetylcholine receptor-rich membranes prepared from Torpedo marmorata electroplax according to the method of Cohen et al. (Cohen, J.B., Weber, M., Huchet, M. and Changeux, J.-P. (1972) FEBS Lett. 26, 43--27). 2. The ESR spectra of the androstanol spin label display a component corresponding to molecules which are immobilized with respect to rotation about the long molecular axis (tau R greater than or equal to 50 ns), in addition to the fluid lipid bilayer component in which the molecules are rotating rapidly about their long axes (tau R approximately 1 ns). This immobilized component is observed throughout the temperature range 2--22 degrees C, at an approximately constant relative intensity of approx. 45% of the total, which is quantitatively the same as previously observed with fatty acid spin labels.

Androstanols↗
PubMed1998

Androstane metabolites bind to and deactivate the nuclear receptor CAR-beta.

The orphan receptor CAR-beta binds DNA as a heterodimer with the retinoid-X receptor and activates gene transcription in a constitutive manner. Here we show that, in contrast to the classical nuclear receptors, the constitutive activity of CAR-beta results from a ligand-independent recruitment of transcriptional co-activators. While searching for potential ligands of CAR-beta, we found that the steroids androstanol and androstenol inhibit the constitutive activity of CAR-beta. This effect is stereospecific: only 3alpha-hydroxy, 5alpha-reduced androstanes are active. These androstanes do not interfere with heterodimerization or DNA binding of CAR-beta; instead, they promote co-activator release from the ligand-binding domain. These androstane ligands are examples of naturally occurring inverse agonists that reverse transcriptional activation by nuclear receptors. CAR-beta (constitutive androstane receptor-beta), therefore, defines an unanticipated steroidal signalling pathway that functions in a manner opposite to that of the conventional nuclear receptor pathways.

Androstanes↗
PubMed2006

CAR2 displays unique ligand binding and RXRalpha heterodimerization characteristics.

The constitutive androstane receptor (CAR; NR1I3) regulates the expression of genes involved in xenobiotic metabolism. Alternative splicing of the human CAR gene yields an array of mRNAs that encode structurally diverse proteins. One form of CAR, termed CAR2, contains an additional four amino acids (SPTV) that are predicted to reshape the ligand-binding pocket. The current studies show a marked, ligand-independent, CAR2-mediated transactivation of reporters containing optimal DR-3, DR-4, and DR-5 response elements, and reporters derived from the natural CYP2B6 and CYP3A4 gene promoters. Overexpression of the RXRalpha ligand binding domain was critical for achieving these effects. CAR2 interaction with SRC-1 was similarly dependent on the coexpression of RXRalpha. Mutagenesis of Ser233 (SPTV) to an alanine residue yielded a receptor possessing higher constitutive activity. Alternatively, mutating Ser233 to an aspartate residue drastically reduced the transactivation capacity of CAR2. The respective abilities of these mutagenized forms of CAR2 to transactivate a DR-4 x 3 reporter element correlated with their ability to interact with RxRalpha and to recruit SRC-1 in a ligand-regulated manner. Together, these results demonstrate a robust RXRalpha-dependent recruitment of coactivators and transactivation by CAR2. In addition, CAR2 displays novel dose responses to clotrimazole and androstanol compared with the reference form of the receptor while at the same time retaining the ability to bind CITCO. This result supports a hypothesis whereby the four-amino-acid insertion in CAR2 structurally modifies its ligand binding pocket, suggesting that CAR2 is regulated by a set of ligands distinct from those governing the activity of reference CAR.

Androstanols↗
PubMed2002

Modulation of acetaminophen-induced hepatotoxicity by the xenobiotic receptor CAR.

We have identified the xenobiotic receptor CAR (constitutive androstane receptor) as a key regulator of acetaminophen metabolism and hepatotoxicity. Known CAR activators as well as high doses of acetaminophen induced expression of three acetaminophen-metabolizing enzymes in wild-type but not in CAR null mice, and the CAR null mice were resistant to acetaminophen toxicity. Inhibition of CAR activity by administration of the inverse agonist ligand androstanol 1 hour after acetaminophen treatment blocked hepatotoxicity in wild type but not in CAR null mice. These results suggest an innovative therapeutic approach for treating the adverse effects of acetaminophen and potentially other hepatotoxic agents.

Acetaminophen↗
PubMed2005

Antagonist- and inverse agonist-driven interactions of the vitamin D receptor and the constitutive androstane receptor with corepressor protein.

Ligand-dependent signal transduction by nuclear receptors (NRs) includes dynamic exchanges of coactivator (CoA) and corepressor (CoR) proteins. Here we focused on the structural determinants of the antagonist- and inverse agonist-enhanced interaction of the endocrine NR vitamin D receptor (VDR) and the adopted orphan NR constitutive androstane receptor (CAR) from two species with the CoR NR corepressor. We found that the pure VDR antagonist ZK168281 and the human CAR inverse agonist clotrimazole are both effective inhibitors of the CoA interaction of their respective receptors, whereas ZK168281 resembled more the mouse CAR inverse agonist androstanol in its ability to recruit CoR proteins. Molecular dynamics simulations resulted in comparable models for the CoR receptor interaction domain peptide bound to VDR/antagonist or CAR/inverse agonist complexes. A salt bridge between the CoR and a conserved lysine in helix 4 of the NR is central to this interaction, but also helix 12 was stabilized by direct contacts with residues of the CoR. Fixation of helix 12 in the antagonistic/inverse agonistic conformation prevents an energetically unfavorable free floatation of the C terminus. The comparable molecular mechanisms that explain the similar functional profile of antagonist and inverse agonists are likely to be extended from VDR and CAR to other members of the NR superfamily and may lead to the design of even more effective ligands.

Amino Acid Sequence↗
PubMed2005

Role of nuclear receptor CAR in carbon tetrachloride-induced hepatotoxicity.

AIM: To investigate the precise roles of CAR in CCl(4)-induced acute hepatotoxicity. METHODS: To prepare an acute liver injury model, CCl(4) was intraperitoneally injected in CAR+/+ and CAR-/- mice. RESULTS: Elevation of serum alanine aminotransferase and extension of centrilobular necrosis were slightly inhibited in CAR-/- mice compared to CAR+/+ mice without PB. Administration of a CAR inducer, PB, revealed that CCl(4)-induced liver toxicity was partially inhibited in CAR-/- mice compared with CAR+/+ mice. On the other hand, androstanol, an inverse agonist ligand, inhibited hepatotoxicity in CAR+/+ but not in CAR-/- mice. Thus, CAR activation caused CCl(4) hepatotoxicity while CAR inhibition resulted in partial protection against CCl(4)-induced hepatotoxicity. There were no differences in the expression of CYP2E1, the main metabolizing enzyme for CCl(4), between CAR+/+ and CAR-/- mice. However, the expression of other CCl(4)-metabolizing enzymes, such as CYP2B10 and 3A11, was induced by PB in CAR+/+ but not in CAR-/- mice. Although the main pathway of CCl(4)-induced acute liver injury is mediated by CYP2E1, CAR modulates its pathway via induction of CYP2B10 and 3A11 in the presence of activator or inhibitor. CONCLUSION: The nuclear receptor CAR modulates CCl(4)-induced liver injury via induction of CCl(4)-metabolizing enzymes in the presence of an activator. Our results suggest that drugs interacting with nuclear receptors such as PB might play critical roles in drug-induced liver injury or drug-drug interaction even though such drugs themselves are not hepatotoxic.

Androstanols↗
PubMed2002

Orphan nuclear receptor binding site in the human inducible nitric oxide synthase promoter mediates responsiveness to steroid and xenobiotic ligands.

Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are members of the nuclear receptor superfamily that regulate target gene transcription in a ligand-dependent manner. CAR and PXR have a rather broad, overlapping set of ligands that range from natural steroids to xenobiotics and also recognize similar DNA binding sites, referred to as response elements (REs), primarily in promoter regions of cytochrome P450 (CYP) genes. In this study, a CAR and PXR RE, composed of a direct repeat of two GGTTCA motifs in a distance of 4 nucleotides (DR4), was identified in the promoter of the human inducible nitric oxide (NO) synthase (iNOS) gene, which is the first nuclear receptor binding site reported for this promoter. In a heterologous promoter context, the DR4-type sequence also acts as a functional RE for the nuclear receptors for 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25OH2D3) and 3,5,3'-triiodothyronine (T3), VDR and T3R. However, in a direct competition of CAR, PXR, VDR, and T3R, the PXR-retinoid X receptor (RXR) complex appears to be the dominant regulator on the iNOS DR4-type RE. In the natural iNOS promoter context, the DR4-type RE specifically mediates downregulation of promoter activity by the testosterone metabolite androstanol through CAR-RXR heterodimers and upregulation by the xenobiotic drug clotrimazole through PXR-RXR heterodimers. These results were confirmed on the level of mRNA expression. Since an iNOS-induced production of NO is known to influence inflammation and apoptosis, a CAR- and PXR-regulated iNOS activity may explain a modulatory effect of steroids and xenobiotics on these cellular processes.

Androstanols↗
PubMed1994

Lipid-protein interactions and protein dynamics in vesicles containing the nicotinic acetylcholine receptor: a study with ethanol.

Electron paramagnetic resonance (EPR) spectroscopy was used to study the action of ethanol on the protein side chain motions of the nicotinic acetylcholine receptor (nAcChoR) in alkaline extracted membranes from Torpedo nobiliana. EPR spectra of the nAcChoR derivatized with maleimide spin label contain both strongly and weakly immobilized components. The rotational correlation time of the strongly immobilized component decreases by a factor of 2-3-fold with the addition of 1.6 M ethanol, while that of the weakly immobilized component is not significantly altered. EPR spectroscopy was also used to probe the lipid environment immediately surrounding the nAcChoR with stearic acid and phosphatidylcholine spin labeled at the fourteenth acyl carbons (14-SASL and 14-PCSL, respectively), and the steroid spin label androstanol (ASL). EPR spectra of these probes reveal a component corresponding to lipids that are motionally restricted by the receptor (annular lipids) in addition to a more fluid component arising from bulk lipid. Using spectral subtraction, the order of selectivity of these spin labels for the nAcChoR was determined to be ASL > or = 14-SASL > 14-PCSL. The estimated rotational correlation times of the high affinity 14-SASL and ASL probes ranged from approx. 20 to 35 ns. The correlation times of the lower affinity 14-PCSL were generally shorter than those for 14-SASL and ASL and ranged from about 10 to 25 ns. The addition of up to 0.9 M ethanol altered neither the affinity nor the mobility of the motionally restricted EPR component. This suggests that ethanol's actions on the nAcChoR are not mediated via changes at the lipid/protein interface near the center of the bilayer.

Animals↗
PubMed1985

Comparative study of the lipid dynamics in the surface layer of porcine and human high density lipoprotein subclasses by spin labeling.

In order to obtain information on the determinants of the lipid dynamics in the high density lipoproteins (HDL), we have compared the structural properties of human HDL subclasses with porcine HDL artificially subdivided into density subfractions corresponding to those of human HDL. Four different positional isomers of spin labeled fatty acids and spin labeled androstanol experienced more restricted motion in porcine HDL than in the human HDL2 and HDL3 subclasses. The differences in the spin label motion could not be accounted for on the basis of the differences in the chemical composition of the lipoproteins examined. They are, however, most probably due to the specific properties of the interactions between lipids and proteins that differ among the lipoproteins.

Animals↗
PubMed1978

Comparison of large unilamellar vesicles prepared by a petroleum ether vaporization method with multilamellar vesicles: ESR, diffusion and entrapment analyses.

Large unilamellar vesicles, prepared by a petroleum ether vaporization method, were compared to multilamellar vesicles with respect to a number of physical and functional properties. Rotational correlation time approximations, derived from ESR spectra of both hydrophilic (3-doxyl cholestane) and hydrophobic (3-doxyl androstanol) steroid spin probes, indicated similar molecular packing of lipids in bilayers of multilamellar and large unilamellar liposomes. Light scattering measurements demonstrated a reduction in apparent absorbance of large unilamellar vesicles, suggesting loss of multilamellar structure which was confirmed by electron microscopy. Furthermore, large unilamellar vesicles exhibited enhanced passive diffusion rates of small solutes, releasing a greater percentage of their contents within 90 min than multilamellar vesicles, and reflecting the less restricted diffusion of a unilamellar system. The volume trapping capacity of large unilamellar vesicles far exceeded that of multilamellar liposomes, except in the presence of a trapped protein, soy bean trypsin inhibitor, which reduced the volume of the aqueous compartments of large unilamellar vesicles. Finally, measurement of vesicle diameters from electron micrographs of large unilamellar vesicles showed a vesicle size distribution predominantly in the range of 0.1--0.4 micron with a mean diameter of 0.21 micron.

Chromates↗
PubMed2001

Membrane insertion and lipid-protein interactions of bovine seminal plasma protein PDC-109 investigated by spin-label electron spin resonance spectroscopy.

The interaction of the major acidic bovine seminal plasma protein, PDC-109, with dimyristoylphosphatidylcholine (DMPC) membranes has been investigated by spin-label electron spin resonance spectroscopy. Studies employing phosphatidylcholine spin labels, bearing the spin labels at different positions along the sn-2 acyl chain indicate that the protein penetrates into the hydrophobic interior of the membrane and interacts with the lipid acyl chains up to the 14th C atom. Binding of PDC-109 at high protein/lipid ratios (PDC-109:DMPC = 1:2, w/w) results in a considerable decrease in the chain segmental mobility of the lipid as seen by spin-label electron spin resonance spectroscopy. A further interesting new observation is that, at high concentrations, PDC-109 is capable of (partially) solubilizing DMPC bilayers. The selectivity of PDC-109 in its interaction with membrane lipids was investigated by using different spin-labeled phospholipid and steroid probes in the DMPC host membrane. These studies indicate that the protein exhibits highest selectivity for the choline phospholipids phosphatidylcholine and sphingomyelin under physiological conditions of pH and ionic strength. The selectivity for different lipids is in the following order: phosphatidylcholine approximately sphingomyelin > or = phosphatidic acid (pH 6.0) > phosphatidylglycerol approximately phosphatidylserine approximately and rostanol > phosphatidylethanolamine > or = N-acyl phosphatidylethanolamine >> cholestane. Thus, the lipids bearing the phosphocholine moiety in the headgroup are clearly the lipids most strongly recognized by PDC-109. However, these studies demonstrate that this protein also recognizes other lipids such as phosphatidylglycerol and the sterol androstanol, albeit with somewhat reduced affinity.

Animals↗
PubMed2001

Opposite effects of substance P fragments C (anxiogenic) and N (anxiolytic) injected into dorsal periaqueductal gray.

Recent findings implicating neurokinins in the expression of anxiety-like behaviors have stimulated interest in the participation of these neuropeptides in the dorsal periaqueductal gray matter (dPAG), one of the main output regions of the brainstem for the expression of defense reaction. Studies on the behavior of rats submitted to the elevated plus-maze test in this laboratory have shown that microinjections of substance P into the dorsal periaqueductal gray produce anxiogenic-like effects. Now, we analyze what portion of the molecule of substance P is responsible for these effects through the examination of the action of its C- and N-terminus fragments (6-11 and 1-7) in the elevated plus-maze. We also investigated whether these effects are influenced by prior treatment with the tachykinin NK(1) receptor antagonist 17-beta-hydroxy-17-alpha-ethynyl-5alpha-androstanol[3,2-b]pyrimido[1,2-a]benzimidazole (WIN51,708). To this end, rats were implanted with a cannula in the dorsal periaqueductal gray and injected 1 week later with equimolar doses (17.5 and 35 pmol) of either C- or N-fragments of substance P and tested in the elevated plus-maze. The results show that the C-terminal fragment has an anxiogenic profile of effects, including reduction in the number of entries and time spent in the open arms of the maze, plus increases in scanning, stretched-attend posture, head dipping and flat-back approach. On the other hand, the N-terminal fragment produced opposite effects, namely, an increase in the number of entries and time spent in the open arms of the maze accompanied by an increase in end-arm activity, rearing and head dipping. The tachykinin NK(1) receptor antagonist WIN51,708 (20 mg/kg, i.p.) inhibited the effects of the carboxy-terminal of substance P while it did not change the effects of the N-terminal fragment. Microinjection of WIN51,708 (20 mg/kg, i.p.), by its own, did not produce any significant effects. Therefore, the results indicate that the anxiogenic effects of substance P injected into the dorsal periaqueductal gray are encoded by its carboxy-terminal sequence and due to its action on tachykinin NK(1) receptors.

Analysis of Variance↗