Interaction of the alpha(1B)-adrenergic receptor with gC1q-R, a multifunctional protein.

gC1q-R, a multifunctional protein, was found to bind with the carboxyl-terminal cytoplasmic domain of the alpha(1B)-adrenergic receptor (173 amino acids, amino acids 344-516) in a yeast two-hybrid screen of a cDNA library prepared from the rat liver. In a series of studies with deletion mutants in this region, the ten arginine-rich amino acids (amino acids 369-378) were identified as the site of interaction. The interaction was confirmed by specific co-immunoprecipitation of gC1q-R with full-length alpha(1B)-adrenergic receptors expressed on transfected COS-7 cells, as well as by fluorescence confocal laser scanning microscopy, which showed co-localization of these proteins in intact cells. Interestingly, the alpha(1B)-adrenergic receptors were exclusively localized to the region of the plasma membrane in COS-7 cells that expressed the alpha(1B)-adrenergic receptor alone, whereas gC1q-R was localized in the cytoplasm in COS-7 cells that expressed gC1q-R alone; however, in cells that co-expressed alpha(1B)-adrenergic receptors and gC1q-R, most of the alpha(1B)-adrenergic receptors were co-localized with gC1q-R in the intracellular region, and a remarkable down-regulation of receptor expression was observed. These observations suggest a new role for the previously identified complement regulatory molecule, gC1q-R, in regulating the cellular localization and expression of the alpha(1B)-adrenergic receptors.     

Molecular cloning and expression of the cDNA for a novel alpha 1-adrenergic receptor subtype

A novel alpha 1-adrenergic receptor subtype has been cloned from a bovine brain cDNA library. The deduced amino acid sequence is that of a 466-residue polypeptide. The structure is similar to that of the other adrenergic receptors as well as the larger family of G protein-coupled receptors that have a presumed seven-membrane-spanning domain topography. The greatest sequence identity of this receptor protein is with the previously cloned hamster alpha 1B-adrenergic receptor being approximately 72% within the presumed membrane-spanning domains. Localization on different human chromosomes provides evidence that the bovine cDNA is distinct from the hamster alpha 1B-adrenergic receptor. The bovine cDNA clone expressed in COS7 cells revealed 10-fold higher affinity for the alpha 1-adrenergic antagonists WB4101 and phentolamine and the agonist oxymetazoline as compared with the alpha 1B receptor, results similar to pharmacologic binding properties described for the alpha 1A receptor. Despite these similarities in pharmacological profiles, the bovine alpha 1-adrenergic receptor is sensitive to inhibition by the alkylating agent chloroethylclonidine unlike the alpha 1A-adrenergic receptor subtype. In addition, a lack of expression in tissues where the alpha 1A subtype exists suggests that this receptor may actually represent a novel alpha 1-adrenergic receptor subtype not previously appreciated by pharmacological criteria.     

alpha 2B-Adrenoceptor levels govern agonist and inverse agonist responses in PC12 cells

Receptor density is an important determinant of cellular effector responses to receptor activation. We analysed cytosolic Ca(2+) responses to alpha(2)-adrenergic agents in PC12 cells expressing human alpha(2B)-adrenergic receptors (AR) at two densities (3.8 and 1.3 pmol/mg protein). The efficacy (E(max)) of agonists was greater in cells with higher receptor expression; while the potency (EC(50)) of norepinephrine and oxymetazoline was independent of alpha(2B)-AR levels. Several classical alpha(2)-AR antagonists behaved as either partial or inverse agonists in a receptor density-dependent fashion. No apparent structural similarities were found among the inverse agonists, precluding simple predictions of inverse agonist activity. Transfected PC12 cells expressing alpha(2B)-AR at relatively high density would be a useful approach to screen inverse agonists for this class of receptors. Our results further indicate that receptor density significantly influences the properties of ligands, not only of partial agonists as predicted by classical receptor theory, but also of antagonists and full agonists.     

Na+/H+ exchangers, alpha-2-adrenergic receptors, sodium sensitivity and arterial hypertension]

Existing evidences indicate that a crossed regulation between alpha 2-adrenergic receptors and Na+/H+ exchanger(s) exists, that Na decreases the affinity of alpha 2-adrenergic receptors for agonists and antagonists, that intracellular Na+ and H+ ion concentrations regulate Na+/H+ exchanger activity, that intracellular pH controls the affinity of the alpha 2-adrenergic receptors for their agonists and antagonists. Alterations of alpha 2-adrenergic receptor densities and allosteric regulation by sodium have been demonstrated in sodium-dependent hypertension in rats. Increased Na+/H+ exchanger activity has been reported in genetic hypertension. Nevertheless, cosegregation experiments and human genetic polymorphism suggest that the exchanger could not be related to hypertension. We propose the following hypothesis: the increased Na+/H+ exchanger characteristic of hypertension could be secondary to the abnormalities of the alpha 2-adrenergic receptors found in hypertension, probably through the alteration of the sodium allosteric effect on these receptors.     

Interaction of the ubiquitin carboxyl terminal esterase L1 with α2-adrenergic receptors inhibits agonist-mediated p44/42 MAP kinase activation

Neuroprotective effects of α₂-adrenergic receptor (AR) agonists are mediated via the α_2AAR subtype, but the molecular mechanisms underlying these actions are still not elucidated. A two-hybrid screen was performed to identify new proteins that may control α₂AR receptor function and trafficking. This screen identified the ubiquitin carboxyl-terminal hydrolase-L1 (Uch-L1), a protein associated with Parkinson's disease, as α₂AR interacting protein. This interaction was confirmed and evaluated by GST pull down assays demonstrating that Uch-L1 binds preferentially to the α_2AAR subtype and only with less affinity to α_2BAR and α_2CAR. Co-immunoprecipitation of epitope-tagged proteins confirmed the specificity of this interaction in vivo. Moreover, co-transfection of a truncated G-protein coupled receptor kinase-DNA preventing α₂AR phosphorylation led to an increased signal-strength of coimmunoprecipitated Uch-L1. Confocal laser microscopy showed that interaction of α_2AAR and Uch-L1 occurred in the cytoplasm. α₂AR agonist mediated activation of p44/42 MAP Kinase was drastically decreased in the presence of Uch-L1 indicating a functional relevance of this interaction. These findings may present a mechanism contributing to subtype-specific α₂AR trafficking and a potential pathway for the neuroprotective effects of α₂AR agonists.     

The Golgi-associated PDZ Domain Protein PIST/GOPC Stabilizes the β1-Adrenergic Receptor in Intracellular Compartments after Internalization

Many G-protein-coupled receptors carry C-terminal ligand motifs for PSD-95/discs large/ZO-1 (PDZ) domains; via interaction with PDZ domain-containing scaffold proteins, this allows for integration of receptors into signaling complexes. However, the presence of PDZ domain proteins attached to intracellular membranes suggests that PDZ-type interactions may also contribute to subcellular sorting of receptors. The protein interacting specifically with Tc10 (PIST; also known as GOPC) is a trans-Golgi-associated protein that interacts through its single PDZ domain with a variety of cell surface receptors. Here we show that PIST controls trafficking of the interacting β1-adrenergic receptor both in the anterograde, biosynthetic pathway and during postendocytic recycling. Overexpression and knockdown experiments show that PIST leads to retention of the receptor in the trans-Golgi network (TGN), to the effect that overexpressed PIST reduces activation of the MAPK pathway by β1-adrenergic receptor (β1AR) agonists. Receptors can be released from retention in the TGN by coexpression of the plasma membrane-associated scaffold PSD-95, which allows for transport of receptors to the plasma membrane. Stimulation of β1 receptors and activation of the cAMP pathway lead to relocation of PIST from the TGN to an endosome-like compartment. Here PIST colocalizes with SNX1 and the internalized β1AR and protects endocytosed receptors from lysosomal degradation. In agreement, β1AR levels are decreased in hippocampi of PIST-deficient mice. Our data suggest that PIST contributes to the fine-tuning of β1AR sorting both during biosynthetic and postendocytic trafficking.     

The alpha2A-adrenergic receptor discriminates between Gi heterotrimers of different betagamma subunit composition in Sf9 insect cell membranes

In view of the expanding roles of the betagamma subunits of the G proteins in signaling, the possibility was raised that the rich diversity of betagamma subunit combinations might contribute to the specificity of signaling at the level of the receptor. To test this possibility, Sf9 cell membranes expressing the recombinant alpha2A-adrenergic receptor were used to assess the contribution of the betagamma subunit composition. Reconstituted coupling between the receptor and heterotrimeric Gi protein was assayed by high affinity, guanine nucleotide-sensitive binding of the alpha2-adrenergic agonist, [3H]UK-14,304. Supporting this hypothesis, the present study showed clear differences in the abilities of the various betagamma dimers, including those containing the beta3 subtype and the newly described gamma4, gamma10, and gamma11 subtypes, to promote interaction of the same alphai subunit with the alpha2A-adrenergic receptor.     

Regulation of α(2B)-adrenergic receptor-mediated extracellular signal-regulated kinase 1/2 (ERK1/2) activation by ADP-ribosylation factor 1

A number of signaling molecules are involved in the activation of the mitogen-activated protein kinase (MAPK) pathway by G protein-coupled receptors. In this study, we have demonstrated that α(2B)-adrenergic receptor (α(2B)-AR) interacts with ADP-ribosylation factor 1 (ARF1), a small GTPase involved in vesicle-mediated trafficking, in an agonist activation-dependent manner and that the interaction is mediated through a unique double Trp motif in the third intracellular loop of the receptor. Interestingly, mutation of the double Trp motif and siRNA-mediated depletion of ARF1 attenuate α(2B)-AR-mediated activation of extracellular signal-regulated kinases 1/2 (ERK1/2) without altering receptor intracellular trafficking, whereas expression of the constitutively active mutant ARF1Q71L and ARNO, a GDP-GTP exchange factor of ARF1, markedly enhances the activation of Raf1, MEK1, and ERK1/2. These data strongly demonstrate that the small GTPase ARF1 modulates ERK1/2 activation by α(2B)-AR and provide the first evidence indicating a novel function for ARF1 in regulating the MAPK signaling pathway.     

Selective inhibition of alpha1A-adrenergic receptor signaling by RGS2 association with the receptor third intracellular loop

Regulators of G-protein signaling (RGS) proteins act directly on Galpha subunits to increase the rate of GTP hydrolysis and to terminate signaling. However, the mechanisms involved in determining their specificities of action in cells remain unclear. Recent evidence has raised the possibility that RGS proteins may interact directly with G-protein-coupled receptors to modulate their activity. By using biochemical, fluorescent imaging, and functional approaches, we found that RGS2 binds directly and selectively to the third intracellular loop of the alpha1A-adrenergic receptor (AR) in vitro, and is recruited by the unstimulated alpha1A-AR to the plasma membrane in cells to inhibit receptor and Gq/11 signaling. This interaction was specific, because RGS2 did not interact with the highly homologous alpha1B- or alpha1D-ARs, and the closely related RGS16 did not interact with any alpha1-ARs. The N terminus of RGS2 was required for association with alpha1A-ARs and inhibition of signaling, and amino acids Lys219, Ser220, and Arg238 within the alpha1A-AR i3 loop were found to be essential for this interaction. These findings demonstrate that certain RGS proteins can directly interact with preferred G-protein-coupled receptors to modulate their signaling with a high degree of specificity.     

RACK1 regulates the cell surface expression of the G protein-coupled receptor for thromboxane A(2).

We used the yeast two-hybrid system to screen for proteins that interact with the C-terminus of the beta isoform of the thromboxane A(2) receptor (TPbeta). This screen identified receptor for activated C-kinase 1 (RACK1) as a new TPbeta-interacting protein. Here, we show that RACK1 directly binds to the C-terminus and the first intracellular loop of TPbeta. The TPbeta-RACK1 association was further confirmed by co-immunoprecipitation studies in HEK293 cells and was not modulated by stimulation of the receptor. We observed that cell surface expression of TPbeta was increased when RACK1 was overexpressed, while it was inhibited when endogenous RACK1 expression was knocked down by small interfering RNA. Confocal microscopy confirmed the impaired cell surface expression of TPbeta and suggested that the receptors remained predominantly localized in the endoplasmic reticulum (ER) in RACK1-depleted cells. Confocal microscopy also revealed that a transient TPbeta-RACK1 association takes place in the ER. The effect of RACK1 on receptor trafficking to the cell surface appears to be selective to some G protein-coupled receptors (GPCRs) because inhibition of RACK1 expression also affected cell surface targeting of the angiotensin II type 1 receptor and CXCR4 but not of beta(2)-adrenergic and prostanoid DP receptors. Our data demonstrate for the first time a direct interaction between RACK1 and a GPCR and identify a novel role for RACK1 in the regulation of the transport of a membrane receptor from the ER to the cell surface.     

Inhibition of the lipolytic action of beta-adrenergic agonists in human adipocytes by alpha-adrenergic agonists

The aim of this study was to define the role of the alpha-adrenergic receptor in the regulation of lipolysis by human adipocytes. Glycerol production by isolated human adipocytes was stimulated by the pure beta-adrenergic agonist isoproterenol in a dose-dependent fashion. This stimulation of lipolysis was inhibited by the alpha-adrenergic agonists methoxamine, phenylephrine, and clonidine. Epinephrine-stimulated lipolysis was potentiated by the alpha-adrenergic antagonists, dihydroergocryptine, phentolamine, phenoxybenzamine, and yohimbine. Whereas the attenuation of beta-adrenergic agonist-stimulated lipolysis by alpha-adrenergic agonists was reversed completely by the alpha 2-adrenergic antagonist yohimbine, the alpha 1-antagonist prazosin did not reverse such attenuation. It is concluded that alpha-adrenergic agonists act as antilipolytic agents in human adipocytes and that this action may result from the interaction of these compounds with a population of alpha 2-adrenergic receptors.     

Molecular basis of inverse agonism in a G protein-coupled receptor

G protein-coupled receptors (GPCRs) recognize a wide variety of extracellular ligands to control diverse physiological processes. Compounds that bind to such receptors can either stimulate, fully or partially (full or partial agonists), or reduce (inverse agonists) the receptors' basal activity and receptor-mediated signaling. Various studies have shown that the activation of receptors through binding of agonists proceeds by conformational changes as the receptor switches from a resting to an active state leading to G protein signaling. Yet the molecular basis for differences between agonists and inverse agonists is unclear. These different classes of compounds are assumed to switch the receptors' conformation in distinct ways. It is not known, however, whether such switching occurs along a linear 'on-off' scale or whether agonists and inverse agonists induce different switch mechanisms. Using a fluorescence-based approach to study the alpha2A-adrenergic receptor (alpha(2A)AR), we show that inverse agonists are differentiated from agonists in that they trigger a very distinct mode of a receptor's switch. This switch couples inverse agonist binding to the suppression of activity in the receptor.     

Molecular cloning and expression of the cDNA for the alpha 1A-adrenergic receptor. The gene for which is located on human chromosome 5.

Pharmacological and molecular cloning studies have demonstrated heterogeneity of alpha 1-adrenergic receptors. We have now cloned two alpha 1-adrenergic receptors from a rat cerebral cortex cDNA library, using the hamster alpha 1B-adrenergic receptor as a probe. The deduced amino acid sequence of clone RA42 encodes a protein of 560 amino acids whose putative topology is similar to that of the family of G-protein-coupled receptors. The primary structure though most closely resembles that of an alpha 1-adrenergic receptor, having approximately 73% amino acid identity in the putative transmembrane domains with the previously isolated hamster alpha 1B receptor. Analysis of the ligand binding properties of RA42 expressed in COS-7 cells with a variety of adrenergic ligands demonstrates a unique alpha 1-adrenergic receptor pharmacology. High affinity for the antagonist WB4101 and agonists phenylephrine and methoxamine suggests that cDNA RA42 encodes the alpha 1A receptor subtype. Northern blot analysis of various rat tissues also shows the distribution expected of the alpha 1A receptor subtype with abundant expression in vas deferens followed by hippocampus, cerebral cortex, aorta, brainstem, heart and spleen. The second alpha 1-adrenergic receptor cloned represents the rat homolog of the hamster alpha 1B subtype. Expression of mRNA for this receptor is strongly detected in liver followed by heart, cerebral cortex, brain stem, kidney, lung, and spleen. This study provides definitive evidence for the existence of three alpha 1-adrenergic receptor subtypes.     

Hippocampal astrocytes exhibit Ca2+-elevating muscarinic cholinergic and histaminergic receptors in situ

Recent findings suggest that astrocytes respond to neuronally released neurotransmitters with Ca2+ elevations. These Ca2+ elevations may trigger astrocytes to release glutamate, affecting neuronal activity. Neuronal activity is also affected by modulatory neurotransmitters that stimulate G protein-coupled receptors. These neurotransmitters, including acetylcholine and histamine, might affect neuronal activity by triggering Ca2+-dependent release of neurotransmitters from astrocytes. However, there is no physiological evidence for histaminergic or cholinergic receptors on astrocytes in situ. We asked whether astrocytes have these receptors by imaging Ca2+-sensitive dyes sequestered by astrocytes in hippocampal slices. Our results show that immunocytochemically identified astrocytes respond to carbachol and histamine with increases in intracellular free Ca2+ concentration. The H1 histamine receptor antagonist chlorpheniramine inhibited responses to histamine. Similarly, atropine and the M1-selective muscarinic receptor antagonist pirenzepine inhibited carbachol-elicited responses. Astrocyte responses to histamine and carbachol were compared with responses elicited by alpha1-adrenergic and metabotropic glutamate receptor agonists. Individual astrocytes responded to different subsets of receptor agonists. Ca2+ oscillations were the prevalent response pattern only with metabotropic glutamate receptor stimulation. Finally, functional alpha1-adrenergic receptors and muscarinic receptors were not detected before postnatal day 8. Our data show that astrocytes have acetylcholine and histamine receptors coupled to Ca2+. Given that Ca2+ elevations in astrocytes trigger neurotransmitter release, it is possible that these astrocyte receptors modulate neuronal activity.     

Activation of phospholipase C by alpha 1-adrenergic receptors is mediated by the alpha subunits of Gq family.

High efficiency transient transfection of Cos-7 cells was previously used to establish the functional coupling between G alpha q/G alpha 11 and phospholipase C beta 1 (Wu, D., Lee, C-H., Rhee, S. G., and Simon, M. I. (1992) J. Biol. Chem. 267, 1811-1817). Here the same system was used to study the functional coupling between other guanine nucleotide-binding regulatory protein (G-protein) alpha subunits and phospholipases and to study which G alpha subunits mediate the activation of phospholipase C by the alpha 1-adrenergic receptor subtypes, alpha 1 A, alpha 1 B, and alpha 1 C. We found that G alpha 14 and G alpha 16 behaved like G alpha 11 or G alpha q, i.e. they could activate endogenous phospholipases in Cos-7 cells in the presence of AIFn. The synergistic increase in inositol phosphate release in Cos-7 cells after they were cotransfected with cDNAs encoding G alpha subunits and phospholipase C beta 1 indicates that both G alpha 16 and G alpha 14 can activate phospholipase C beta 1. The activation of phospholipase C beta 1 was restricted to members of the Gq subfamily of alpha subunits. They activated phospholipase C beta 1 but not phospholipase C gamma 1, gamma 2, or phospholipase C delta 3. The cotransfection of Cos-7 cells with cDNAs encoding three different alpha 1-adrenergic receptors and G alpha q or G alpha 11 leads to an increase in norepinephrine-dependent inositol phosphate release. This indicates that G alpha q or G alpha 11 can mediate the activation of phospholipase C by all three subtypes of alpha 1-adrenergic receptors. With the same assay system, G alpha 16 and G alpha 14 appear to be differentially involved in the activation of phospholipase C by the alpha 1-adrenergic receptors. The alpha 1 B subtype receptor gave a ligand-mediated synergistic response in the cells cotransfected with either G alpha 14 or G alpha 16. However, the alpha 1 C receptor responded in cells cotransfected with G alpha 14 but not G alpha 16, and the alpha 1 A receptor showed little synergistic response in cells transfected with either G alpha 14 or G alpha 16. The ability of the alpha 1 A and alpha 1 C receptors to activate phospholipase C through G alpha q and G alpha 11 was also demonstrated in a cell-free system.(ABSTRACT TRUNCATED AT 400 WORDS)     

Two peptides from the alpha 2A-adrenergic receptor alter receptor G protein coupling by distinct mechanisms.

Peptides derived from various regions of the alpha 2A-adrenergic receptor (alpha 2A-AR) were used to study receptor-G protein interactions. Binding of the partial agonist [125I]-p-iodoclonidine and the full agonist [3H]bromoxidine (UK14,304) to membrane preparations from human platelet was potently reduced by peptides (12-14 amino acids) from the second cytoplasmic loop (A) and the C-terminal side of the third cytoplasmic loop (Q). Binding of the antagonist [3H]yohimbine was significantly less affected. Five other peptides had no significant effects on ligand binding at concentrations less than 100 microM. The IC50 values for peptides A and Q were 7 and 27 microM for [125I]-p-iodoclonidine binding at the platelet alpha 2A receptor, 15 and 71 microM for the neuroblastoma-glioma (NG108-15) alpha 2B receptor, and greater than 300 microM for yohimbine binding at both alpha 2A and alpha 2B receptors. Competition studies demonstrate that at concentrations of 100 microM, peptides A and Q reduce the affinity of bromoxidine for the platelet alpha 2A-AR and this effect was abolished in the presence of guanine nucleotide. Alpha 2A-AR-stimulated GTPase activity in platelet membranes was inhibited by peptide Q with an IC50 of 16 microM but A was inactive. These data suggest that both the second cytoplasmic loop and the C-terminal part of the third cytoplasmic loop of the alpha 2A-AR are important in the interaction between the alpha 2-AR and Gi protein. Peptide Q appears to destabilize the high affinity state of the alpha 2-AR by binding directly to Gi thus preventing it from coupling to the receptor under both binding and GTPase assay conditions. The peptide from the second cytoplasmic loop (A) also reduces high affinity agonist binding in a G protein-dependent manner but its interaction with receptor and G protein is distinct in that it does not prevent activation of the G protein. These results provide new information about regions of the alpha 2-adrenergic receptor involved in G protein coupling and high affinity agonist binding.     

Cloning, sequence analysis, and permanent expression of a human alpha 2-adrenergic receptor in Chinese hamster ovary cells. Evidence for independent pathways of receptor coupling to adenylate cyclase attenuation and activation

The gene encoding a human alpha 2-adrenergic receptor was isolated from a human genomic DNA library using a 367-base pair fragment of Drosophila genomic DNA that exhibited 54% identity with the human beta 2-adrenergic receptor and 57% identity with the human alpha 2-adrenergic receptor. The nucleotide sequence of a fragment containing the human alpha 2-receptor gene and 2.076 kilobases of untranslated 5' sequence was determined, and potential upstream regulatory regions were identified. This gene encodes a protein of 450 amino acids and was identified as an alpha 2-adrenergic receptor by homology with published sequences and by pharmacological characterization of the protein expressed in cultured cells. Permanent expression of the alpha 2-receptor was achieved by transfecting Chinese hamster ovary (CHO) cells which lack adrenergic receptors with a 1.5-kilobase NcoI-HindIII fragment of the genomic clone containing the coding region of the gene. The alpha 2-receptor expressed in CHO cells displayed pharmacology characteristic of an alpha 2 A-receptor subtype with a high affinity for yohimbine (Ki = 1 nM) and a low affinity for prazosin (Ki = 10,000 nM). Agonists displayed a rank order of potency in radioligand binding assays of para-aminoclonidine greater than or equal to UK-14304 greater than (-)-epinephrine greater than (-)-norepinephrine greater than (-)-isoproterenol, consistent with the identification of this protein as an alpha 2-receptor. The role of the alpha 2-receptor in modulating intracellular cyclic AMP concentrations was investigated in three transfected cell lines expressing 50, 200, and 1200 fmol of receptor/mg membrane protein. At low concentrations (1-100 nM), (-)-epinephrine attenuated forskolin-stimulated cyclic AMP accumulation by up to 60% in a receptor density-dependent manner. At epinephrine concentrations above 100 nM, cyclic AMP levels were increased up to 140% of the forskolin-stimulated level. Pertussis toxin pretreatment of cells eliminated alpha 2-receptor-mediated attenuation of forskolin-stimulated cyclic AMP levels and enhanced the receptor density-dependent potentiation of forskolin-stimulated cyclic AMP concentrations from 3 to 8-fold. Potentiation of forskolin-stimulated cyclic AMP levels was also elicited by the alpha 2-adrenergic agonists, UK-14304 and para-aminoclonidine, and blocked by the alpha 2-adrenergic antagonist yohimbine, but not by the alpha 1-adrenergic antagonist prazosin or the beta-adrenergic antagonist propranolol.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alpha 1-adrenergic responsiveness of young adult and aged rat submandibular cells in vitro

The present study has evaluated, in vitro, alpha 1-adrenergic receptor mediated responses in submandibular cells from young adult and aged rats. Submandibular glands from different aged rats possess a similar number of alpha 1-adrenergic receptors that display comparable binding characteristics. Following alpha 1-adrenergic stimulation, cells from both groups of rats show a similar ability to mobilize intracellular Ca2+ (45Ca2+ time course, agonist dose-response) and to elicit a functional response (inhibition of protein synthesis by epinephrine) which reflects Ca2+ mobilization.     

Transient high-affinity binding of agonists to alpha 1-adrenergic receptors of intact liver cells

At alpha 1-adrenergic receptors in isolated rat liver parenchymal cells, (-)-epinephrine is potent in eliciting a maximal increase in glycogenolysis (Kact = 24 nM). This contrasts with a 100-fold lower affinity for the agonist at alpha 1-adrenergic receptors of intact hepatocytes determined from equilibrium competition assays with the alpha 1-adrenergic antagonist [3H]prazosin. We demonstrate here that agonists bind to alpha 1-adrenergic receptors of intact liver cells initially with a markedly higher affinity than under equilibrium conditions. When incubations are performed for 15 s at 37 degrees C, the affinity is more than 100-fold higher than that obtained in equilibrium (45 min) assays (IC50 = 28 +/- 3 vs 5300 +/- 400 nM for (-)-epinephrine and 32 +/- 3 vs 6100 +/- 500 nM for (-)-norepinephrine). When incubations are performed at 4 degrees C (150 min), high-affinity binding similar to that obtained in short-term incubations can also be demonstrated. In contrast, antagonist compete with similar affinities in 15 s and 45 min assays, and their dissociation constants are not affected by changes in the incubation temperature. These results indicate that agonists bind to native alpha 1-adrenergic receptors transiently with high affinity. The conversion of receptors to a state of predominantly low affinity for agonists, which occurs rapidly and irreversibly with increasing incubation at 37 degrees C, is inhibited at low incubation temperatures. It is suggested that the high-affinity configuration of the alpha 1-adrenergic receptor for agonists observed in nonequilibrium experiments or at reduced incubation temperatures represents the physiologically relevant state of the alpha 1-adrenergic receptor.