α2-Adrenergic Receptors in Neuronal and Glial Cultures: Characterization and Comparison

Membranes prepared from either neuronal or glial cultures contain alpha 2-adrenergic receptors as determined by the characteristics of [3H]yohimbine [( 3H]YOH) binding. The binding was rapid, reversible, saturable, dependent on the protein concentration used, and reached equilibrium by 5 min in membranes from both neuronal and glial cultures. Scatchard analyses of saturation isotherms revealed similar KD values of 13.7 +/- 1.35 nM (n = 10) for neuronal cultures and 18.42 +/- 2.34 nM (n = 10) for glial cultures. Glial cultures contained many more binding sites for [3H]YOH than neuronal cultures, having a Bmax of 1.6 +/- 0.33 pmol/mg protein (n = 10) compared with 0.143 +/- 0.018 pmol/mg protein (n = 10) in neurons. Drugs selective for alpha 2-adrenergic receptors were the most effective displacers of [3H]YOH binding in both neuronal and glial cultures, i.e., the alpha 2-adrenergic antagonists rauwolscine and yohimbine were better displacers than the other catecholamine antagonists prazosin, corynanthine, or propranolol. The agonists showed the same pattern with the alpha 2-selective drugs clonidine and naphazoline being the most effective competitors for the [3H]YOH site. GTP and its nonhydrolyzable analog. 5'-guanylyl-imidodiphosphate, were able to lower the affinity of the alpha 2-receptors for agonists but not antagonists in membranes from both neuronal and glial cultures, suggesting that the receptors are linked to a G protein in both cell types. The presence of alpha 2-adrenergic receptors in neuronal cultures was also substantiated by light microscopic autoradiography of [3H]YOH binding. In summary, we have demonstrated that both neuronal and glial cultures contain alpha 2-adrenoceptors.     

Characterization of the alpha 1-adrenergic receptor subtype in a smooth muscle cell line

We have identified in the DDT1 smooth muscle cell line a [3H]dihydroergocryptine-binding site having the characteristics of an alpha 1-adrenergic receptor. Specific binding of [3H]dihydroergocryptine to DDT1 cells grown either in monolayer or suspension culture was reversible, saturable, and of high affinity, and the binding site demonstrated stereoselectivity. [3H]Dihydroergocryptine dissociation constants of 1.4 +/- 0.2 nM and 1.4 +/- 0.3 nM were observed for suspension and monolayer cells, respectively. However, the concentration of binding sites in suspension-cultured cells (65,100 +/- 8,300 sites/cell) was significantly greater (p less than 0.001) than that found in monolayer cells (27,900 +/- 4,300 sites/cell). The order of agonist competition for the binding site was epinephrine (Ki = 0.92 +/- 0.32 microM) greater than or equal to norepinephrine (Ki = 2.2 +/- 1.0 microM) greater than isoproterenol (Ki = 137 +/- 17 microM), consistent with an alpha-adrenergic interaction. Results of competition experiments with specific antagonists prazosin (alpha 1-selective) or yohimbine (alpha 2-selective) and a computer modeling technique indicated that the alpha-adrenergic receptor of the DDT1 cell was predominantly (greater than 95%) the alpha 1-subtype.     

Norepinephrine-induced down regulation of alpha 1 adrenergic receptors in cultured rabbit aorta smooth muscle cells

Drug-induced refractoriness of alpha-adrenergic receptor-mediated vasoconstriction may be a clinically important phenomenon. We have investigated the possible molecular mechanisms underlying this phenomenon in cultured vascular smooth muscle cells derived from the rabbit aorta. alpha 1-Adrenergic receptors were identified in membranes prepared from these cells by [125I]HEAT binding. The radioligand bound to a high affinity site (Kd = 140 pM) in a saturable fashion (202 fmol/mg protein). Adrenergic agonists and antagonists competed for binding of [125I]HEAT with the expected order of potency for an alpha 1-receptor, (-)epinephrine greater than or equal to (-) norepinephrine greater than (+)epinephrine greater than isoproterenol and prazosin greater than phentolamine greater than yohimbine. Exposure of cells for 26 hours to 10 microM norepinephrine resulted in a 70% decrease in the number of alpha 1-receptors as measured by [125I]HEAT binding without any significant change in the affinity of the receptor for the ligand. When the alpha-receptors were blocked with 10 microM phentolamine the loss of receptors induced by norepinephrine was completely prevented. Similar down-regulation of the [125I]HEAT binding sites was observed when the alpha 1-agonist phenylephrine was used instead of norepinephrine. It is concluded that alpha-agonists induce down-regulation of aortic smooth muscle alpha 1-receptors. This reduction of alpha-receptors could be important in the mechanisms by which vascular smooth muscle develops refractoriness to alpha-adrenergic stimulation.     

Human apolipoprotein A-II: nucleotide sequence of a cloned cDNA, and localization of its structural gene on human chromosome 1

[3H]yohimbine, a potent and selective alpha 2-adrenergic antagonist was used to label alpha-adrenoceptors in intact human lymphocytes. Binding of [3H]yohimbine was rapid (t1/2 1.5 -2.0 min) and readily reversed by 10 microM phentolamine (t1/2 = 5 - 6 min) and of high affinity (Kd = 3.7 +/- 0.86 nM). At saturation, the total number of binding sites was 19.9 +/- 5.3 fmol/10(7) lymphocytes. Adrenergic agonists competed for [3H]yohimbine binding sites with an order of potency: clonidine greater than (-) epinephrine greater than (-) norepinephrine greater than (+) epinephrine much greater than (-) isoproterenol; adrenergic antagonists with a potency order of yohimbine greater than phentolamine greater than prazosin. These results indicate the presence of alpha 2-adrenoceptors in human lymphocytes.     

Two subpopulations of alpha 1-adrenergic receptors with high and low affinity for agonists: short-term exposure to agonists reduced the high-affinity sites

Short-term receptor regulation by agonists is a well-known phenomenon for a number of receptors, including beta-adrenergic receptors, and has been associated with receptor changes revealed by radioligand binding. In the present study, we investigated the rapid changes in alpha 1-adrenergic receptors induced by agonists. alpha 1-receptors were studied on DDT1 MF-2 smooth muscle cells (DDT1-MF-2 cells) by specific [3H]prazosin binding. In competition binding on membranes and on intact cells at 4 degrees C or at 37 degrees C in 1-min assays, agonists competed for a single class of sites with relatively high affinity. By contrast, in equilibrium binding at 37 degrees C on intact cells agonists competed with two receptor forms (high- and low-affinity). We quantified the receptors in the high-affinity form by measuring the [3H]prazosin binding inhibited by 20 microM norepinephrine (this concentration selectively saturated the high-affinity sites). The low-affinity sites were measured by subtracting the binding of [3H]prazosin to the high-affinity sites from the total specific binding. High-affinity receptors were 85% of the total sites in binding experiments at 4 degrees C, but only 30% at 37 degrees C. On DDT1-MF-2 cells preequilibrated with [3H]prazosin at 4 degrees C, and then shifted to 37 degrees C for a few minutes, norepinephrine selectively reduced the high-affinity sites by 30%. We suggest that at 4 degrees C it is the native form of alpha 1-receptors that is measured, with most of the sites in the high-affinity form, while during incubation at 37 degrees C the norepinephrine present in the binding assay converts most of the receptors to an apparent low-affinity form, so that they are no longer recognized by 20 microM norepinephrine. The nature of this low-affinity form was further investigated. On DDT1-MF-2 cells preincubated with the agonist and then extensively washed at 4 degrees C (to maintain the receptor changes induced by the agonist) the number of receptors recognized by [3H]prazosin at 4 degrees C was reduced by 38%. After fragmentation of the cells, the number of receptors measured at 4 degrees C was the same in control and norepinephrine-treated cells, suggesting that the disruption of cellular integrity might expose the receptors which are probably sequestered after agonist treatment. In conclusion, the appearance of the low affinity for agonists at 37 degrees C may be due to the agonist-induced sequestration of alpha 1-adrenergic receptors, resulting in a limited accessibility to hydrophilic ligands.     

Cyclothiazide binding to functionally active AMPA receptor reveals genuine allosteric interaction with agonist binding sites

The agonist, [3H](-)[S]-1-(2-amino-2-carboxyethyl)-5-fluoro-pyrimidine-2,4-dione ([3H](S)F-Willardiine) binding to functional alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors of resealed plasma membrane vesicles and nerve endings freshly isolated from the rat cerebral cortex displayed two binding sites (K(D1)=33+/-7 nM, B(MAX1)=1.6+/-0.3 pmol/mg protein, K(D2)=720+/-250 nM and B(MAX2)=7.8+/-4.0 pmol/mg protein). The drug which impairs AMPA receptor desensitisation, 6-chloro-3,4-dihydro-3-(2-norbornene-5-yl)-2H-1,2,4-benzothiadiazine-7-sulphonamide-1,1-dioxide (cyclothiazide, CTZ) fully displaced the [3H](S)F-Willardiine binding at a concentration of 500 microM. In the presence of 100 microM CTZ (K(I(CTZ))=60+/-6 microM), both the antagonist [3H]-1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo(F)quinoxaline-7-sulfonamide ([3H]NBQX: K(D)=24+/-4 nM, B(MAX)=12.0+/-0.1 pmol/mg protein) and the high-affinity agonist binding showed similar affinity reduction ([3H](S)F-Willardiine: K(D)=140+/-19 nM, B(MAX)=2.9+/-0.5 pmol/mg protein; [3H]NBQX: K(D)=111+/-34 nM, B(MAX)=12+/-3 pmol/mg protein). To disclose structural correlates underlying genuine allosteric binding interactions, molecular mechanics calculations of CTZ-induced structural changes were performed with the use of PDB data on extracellular GluR2 binding domain dimeric crystals available by now. Hydrogen-bonding and root mean square (rms) values of amino acid residues recognising receptor agonists showed minor alterations in the agonist binding sites itself. Moreover, CTZ binding did not affect dimeric subunit structures significantly. These findings indicated that the structural changes featuring the non-desensitised state could possibly occur to a further site of the extracellular GluR2 binding domain. The increase of agonist efficacy on allosteric CTZ binding may be interpreted in terms of a mechanism involving AMPA receptor desensitisation sequential to activation.     

Calcium binding to extracellular sites of skeletal muscle calcium channels regulates dihydropyridine binding

The binding of dihydropyridine (PN200-110) to skeletal muscle microsomes (which were 84% sealed inside-out vesicles) was not influenced by the addition of calcium or magnesium nor by addition of their chelators (EDTA or EGTA) unless the vesicles were pretreated with the calcium-magnesium ionophore A23187 and EDTA to remove entrapped cations. Separation of inside-out vesicles from right-side-out vesicles by wheat germ agglutinin chromatography revealed that only the right-side-out vesicles exhibited a calcium-, magnesium-, and chelator-dependent binding of PN200-110. Dihydropyridine binding to cardiac sarcolemma membranes (which were 46% inside-out) and to solubilized skeletal muscle membranes was inhibited by EDTA and could be fully restored by 10 microM calcium or 1 mM magnesium. Calcium increased PN200-110 binding to partially purified rabbit skeletal muscle calcium channels from 3.9 pmol/mg protein to 25.5 pmol/mg protein with a pK0.5 = 6.57 +/- 0.059 and a Hill coefficient of 0.56 +/- 0.04. Magnesium increased binding from 0.7 pmol/mg protein to 16.8 pmol/mg protein with a pK0.5 = 3.88 +/- 0.085 and a Hill coefficient of 0.68 +/- 0.074. These studies suggest that calcium binding to high affinity sites or magnesium binding to low affinity sites on the extracellular side of skeletal muscle T-tubule calcium channels regulates dihydropyridine binding. Further, similar calcium and magnesium binding sites exist on the cardiac calcium channel and serve to allosterically regulate dihydropyridine binding.     

Characteristics and metabolism of alpha 1 adrenergic receptors in a nonfusing muscle cell line

The BC3H1 nonfusing muscle cell line possesses binding sites for [3H]prazosin. These binding sites are typically alpha 1 adrenergic receptors as shown by their greater affinity (3700-fold) for prazosin than for yohimbine. Both kinetic and equilibrium analyses indicated that [3H]prazosin interacted with only one category of independent binding sites with the following characteristics. KD = 0.13 +/- 0.01 nM. Bmax = 97 +/- 5 fmol/mg of protein corresponding to 25,000 sites/cell (n = 17). Biosynthesis of the alpha 1 adrenergic receptor was investigated at cell confluency (when the number of cells and their total protein content were constant). Phenoxybenzamine (10(-9) M) irreversibly blocked 50% of the alpha 1 receptors in intact cells. More than 95% blockade of receptors was obtained with 10(-7) M phenoxybenzamine. After this blockade, new alpha 1 adrenergic receptors reappeared in the cells with monoexponential kinetics. These new receptors corresponded to synthesized receptors since their appearance was blocked by cycloheximide (1 micrograms/ml). The cycloheximide action was reversible. If one makes the simple and probable hypotheses that the receptor production is constant and that degradation is a monoexponential process, the analysis of the kinetics of reappearance allows the determination of the rate constant for receptor degradation (k = 0.03 h-1) and the rate of receptor production (r = 3.2 fmol/mg/h) corresponding to the synthesis of about 760 receptors/cell/h. The half-life of the receptor was 23 h.     

Mechanism of vascular relaxation by thaligrisine: functional and binding assays

In the present study we examine the mechanism by which thaligrisine, a bisbenzyltetrahydroisoquinoline alkaloid, inhibits the contractile response of vascular smooth muscle. The work includes functional studies on rat isolated aorta and tail artery precontracted with noradrenaline or KCl. In other experiments rat aorta was precontracted by caffeine in the presence or absence of extracellular Ca2+. In order to assess whether thaligrisine interacts directly with calcium channel binding sites or with alpha-adrenoceptors we examined the effect of the alkaloid on [3H]-(+)-cis diltiazem, [3H]-nitrendipine and [3H]-prazosin binding to cerebral cortical membranes. The functional studies showed that the alkaloid inhibited in a concentration-dependent manner the contractile response induced by depolarization in rat aorta (IC50 = 8.9+/-2.9 microM, n=5) and in tail artery (IC50 = 3.04+/-0.3 microM, n=6) or noradrenaline induced contraction in rat aorta (IC50 = 23.0+/-0.39 microM, n=9) and in tail artery (IC50 = 3.8+/-0.9 microM, n=7). In rat aorta, thaligrisine concentration-dependently inhibited noradrenaline-induced contraction in Ca2+-free solution (IC50 = 13.3 microM, n=18). The alkaloid also relaxed the spontaneous contractile response elicited by extracellular calcium after depletion of noradrenaline-sensitive intracellular stores (IC50 = 7.7 microM, n=4). The radioligand receptor-binding study showed that thaligrisine has higher affinity for [3H]-prazosin than for [3H]-(+)-cis-diltiazem binding sites, with Ki values of 0.048+/-0.007 microM and 1.5+/-1.1 microM respectively. [3H]-nitrendipine binding was not affected by thaligrisine. The present work provides evidence that thaligrisine shows higher affinity for [3H]-prazosin binding site than [3H]-(+)-cis-diltiazem binding sites, in contrast with tetrandrine and isotetrandrine that present similar affinity for both receptors. In functional studies thaligrisine, acted as an alpha1-adrenoceptor antagonist and as a Ca2+ channel blocker, relaxing noradrenaline or KCl-induced contractions in vascular smooth muscle. This compound specifically inhibits the refilling of internal Ca2+-stores sensitive to noradrenaline, by blocking Ca2+-entry through voltage-dependent Ca2+-channels.     

Subcellular distribution of alpha 1-adrenergic receptors in rat liver

The distribution of alpha 1-adrenergic receptors in rat liver subcellular fractions was studied using the alpha 1-adrenergic receptor ligand [3H]prazosin. The highest number of [3H]prazosin binding sites was found in a plasma membrane fraction followed by 2 Golgi and a residual microsomal fraction, the numbers of binding sites were 1145, 845, 629 and 223 fmol/mg protein, respectively. When the binding in these fractions was compared with the activity of plasma membrane 'marker' enzymes in the same fractions a relative enrichment of [3H]prazosin binding sites was found in the residual microsomes and one of the Golgi fractions. Photoaffinity labelling with 125I-arylazidoprazosin in combination with SDS-polyacrylamide gel electrophoresis revealed the specific binding to 40 and 23 kDa entities in a Golgi fraction, while in plasma membranes the binders had an apparent molecular mass of 36 and 23 kDa. When [3H]prazosin was injected in vivo into rat portal blood followed by subcellular fractionation of liver, a pattern of an initial rapid decline and thereafter a slow decline of radioactivity was noted in all fractions. Additionally, in the two Golgi fractions a transient accumulation of radioactivity occurred between 5 and 10 min after the injection. The ED50 values for displacement of [3H]prazosin with adrenaline was lowest in the plasma membrane fraction, followed by the residual microsomes and Golgi fractions, the values were 10(-6), 10(-5) and 10(-4) mol/l, respectively. On the basis of lack of correlation between distribution of alpha 1-adrenergic antagonist binding and adenylate cyclase activity, differences in the molecular mass of alpha 1-adrenergic antagonist binders, differences in the kinetics of in vivo binding and accumulation of [3H]prazosin and also differences in agonist affinity between plasma membrane and Golgi fractions, it is concluded that alpha 1-adrenergic receptors are localized to low-density intracellular membranes involved in receptor biosynthesis and endocytosis.     

Decreased alpha 1-adrenoceptor responsiveness and density in liver cells of thyroidectomized rats

The effects of hypothyroidism on the hepatic alpha 1-receptor system were studied in isolated rat liver cells. Phenylephrine and vasopressin caused concentration-dependent activation of glycogen phosphorylase and release of 45Ca from 45Ca-loaded cells in either normal or thyroidectomized rats. However, the magnitude of both responses to phenylephrine was markedly suppressed after thyroidectomy and could be restored to near normal levels by in vivo treatment with 1-triiodothyronine (0.25 mg/kg/day) for 4 days. The potency of vasopressin to induce phosphorylase activation and 45Ca release was only slightly reduced by thyroidectomy. Binding of [3H]prazosin to putative alpha 1-receptors in purified liver plasma membranes revealed that the above changes were accompanied by a decrease in the density of binding sites from 567 +/- 51 fmol/mg of protein in controls to 326 +/- 51 fmol/mg in thyroidectomized rats and a return to 498 +/- 23 fmol/mg in thyroidectomized rats treated with 1-triiodothyronine. The affinity of binding sites for [3H]prazosin or for alpha-receptor agonists was the same in the three groups of rats and affinity for epinephrine was unaffected by the presence of guanyl-5'-yl imidodiphosphate (30-100 microM). From these findings, it appears that a reduction in the number of hepatic alpha 1-receptors is responsible for the selective decrease in alpha-adrenergic responses in the hypothyroid rat liver. These changes are opposite to those previously reported for hepatic beta-receptors.     

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.     

Binding of endomorphin-2 to mu-opioid receptors in experimental mouse mammary adenocarcinoma.

Endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) binds with high affinity and selectivity to the mu-opioid receptor. In the present study, [125I]endomorphin-2 has been used to characterize mu-opioid-binding sites on transplantable mouse mammary adenocarcinoma cells. Cold saturation experiments performed with [125I]endomorphin-2 (1 nM) show biphasic binding curves in Scatchard coordinates. One component represents high affinity and low capacity (K(d) = 18.79 +/- 1.13 nM, B(max) = 635 +/- 24 fmol/mg protein) and the other shows low affinity and higher capacity (K(d) = 7.67 +/- 0.81 microM, B(max) = 157 +/- 13 pmol/mg protein) binding sites. The rank order of agonists competing for the [125I]endomorphin-2 binding site was [d-1-Nal3]morphiceptin > endomorphin-2 > [d-Phe3]morphiceptin > morphiceptin > [d-1-Nal3]endomorphin-2, indicating binding of these peptides to mu-opioid receptors. The uptake of 131I-labeled peptides administered intraperitoneally to tumor-bearing mice was also investigated. The highest accumulation in the tumor was observed for [d-1-Nal3)morphiceptin, which reached the value of 8.19 +/- 1.14% dose/g tissue.     

Characterization of the binding sites for nimodipine and (-)-desmethoxyverapamil in bovine cardiac sarcolemma

The bovine cardiac sarcolemmal binding sites for the dihydropyridine nimodipine and the phenylalkylamine (-)-desmethoxyverapamil were studied. The density of the nimodipine and (-)-desmethoxyverapamil binding sites increased 8.3-fold and 3.4-fold with the sarcolemma. The binding sites for both compounds were destroyed by trypsin. Nimodipine bound in the presence of 1 mM free calcium to a high-affinity and a low-affinity site with apparent Kd values of 0.35 +/- 0.09 nM (n = 9) and 33 +/- 6.0 nM (n = 9) and with apparent densities of 0.3 +/- 0.05 pmol/mg (n = 9) and 8.2 +/- 1.0 pmol/mg (n = 9). The binding to the high-affinity site was abolished by 1 mM EGTA. The binding sites were specific for dihydropyridines. The (-)-isomers of several phenylalkylamines inhibited nimodipine binding by an apparent allosteric mechanism. (-)-Desmethoxyverapamil bound in the presence of 5 mM EGTA to a high-affinity and a low-affinity site with apparent Kd values of 1.4 +/- 0.3 nM (n = 6) and 171 +/- 26 nM (n = 6) and with apparent densities of 0.16 +/- 0.02 pmol/mg (n = 6) and 13.6 +/- 2.7 pmol/mg (n = 6). The binding to both sites was inhibited by calcium with a half-maximal concentration of 4.3 mM. The binding sites were specific for the other phenylalkylamines and had a higher affinity for the (-)-isomers than for the (+)-isomers. Nimodipine inhibited the binding of (-)-desmethoxyverapamil by an apparent allosteric mechanism. d-cis-Diltiazem inhibited non-competitively the binding of (-)-[3H]desmethoxyverapamil with a Ki of 3.7 microM. Diltiazem up to concentrations of 10 microM did not affect the amount of nimodipine bound at equilibrium at 20 degrees C. However, but in agreement with this result, diltiazem decreased threefold at 20 degrees C the dissociation and association rates for the high-affinity nimodipine receptor. These rates were only marginally affected at 4 degrees C and 37 degrees C. d-cis-Diltiazem reversed in a competitive manner the inhibition of nimodipine binding elicited by the addition of (-)-desmethoxyverapamil with a Ka value of 1.6 microM. The amount of nimodipine bound was inhibited by 50% by the adenosine uptake inhibitors nitrobenzylthioinosine and hexobendine with apparent median inhibitory concentrations of 1 nM and 3 nM, respectively. Nitrobenzylthioinosine completely abolished binding of nimodipine to the low-affinity site, but did not affect binding to the high-affinity site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ontogeny of alpha 1- and beta-adrenergic receptors in rat lung

The binding characteristics of the alpha 1-selective adrenergic ligand [3H]-prazosin were determined in particulate membranes of rat lung from day 18 of gestation to adulthood. Specific binding was present at all ages studied, was reversible and inhibition of specific binding by agonists followed the order of potency: (-)-epinephrine = (-)-norepinephrine much greater than (-)-isoproterenol greater than (+)-norepinephrine. Inhibition by antagonists followed the order of potency: prazosin greater than WB4101, much greater than yohimbine. Binding capacity increased during the neonatal period from 52 +/- 9 fmoles x mg-1 protein in lung preparations on day 18 of a 21 day gestation increasing to 105 +/- 4 fmoles x mg-1 protein (mean +/- SE) by postnatal day 15. Binding activity decreased thereafter, reaching adult levels by 28 days of postnatal age, 62 +/- 3 fmoles x mg-1 protein. This pattern of alpha 1-adrenergic receptor density was distinct from that of beta-adrenergic receptors identified in rat lung membrane with the beta- adrenergic antagonist, (-)-[3H]dihydroalprenolol ((-)-[3H]DHA). (-)-[3H]DHA binding increased dramatically during this same time period, from 46 +/- 4 fmoles x mg-1 protein on day 18 of gestation to 496 +/- 44 fmoles x mg-1 protein in the adult lung. Affinity for [3H]-prazosin and (-)-[3H]DHA did not change with age. Pulmonary alpha 1-adrenergic receptors are present as early as 18 days of gestation in the rat and alpha 1-adrenergic receptor density is maximal by 15 days of postnatal age. The timing of the changes in alpha 1-adrenergic receptors correlates with the timing of increased sympathetic innervation of the developing rat lung and is distinct from that of beta-adrenergic receptor sites.     

Identification and characterization of (3H)-rauwolscine binding to alpha 2-adrenoceptors in the canine saphenous vein

The biochemical exploration of the alpha 2-adrenergic receptors was investigated in the canine saphenous vein using the highly selective alpha 2-adrenergic antagonist rauwolscine as a tritiated ligand. Following an enzymatic digestive pretreatment, we isolated a purified smooth muscle cell membrane fraction from saphenous veins in quantity sufficient to permit us to study the venous alpha 2-adrenoceptor content. The binding of tritiated rauwolscine was rapid, specific, saturable and reversible. The presence of high affinity binding sites (Kd = 1.53 +/- 0.71 nM) with a density of binding Bmax of 125.2 +/- 43.1 fmol/mg protein was demonstrated on a unique class of non interacting sites (nHill = 1.001 +/- 0.06). The kinetically derived Kd was 1.28 nM, in good agreement with the value obtained from saturation isotherms. The pharmacological profile of these sites was assessed by the comparison of the potency of alpha-adrenergic agonists and antagonists to inhibit 1 nM (3H)-rauwolscine. Their efficacy was respectively: rauwolscine greater than phentolamine greater than RX 781094 greater than clonidine much greater than prazosin greater than (-)-phenylephrine greater than (-)-noradrenaline. The results showed that (3H)-rauwolscine bound specifically to sites in our membranal preparation, which had the pharmacological characteristics of the alpha 2-adrenoceptors. The correlation between biochemical and pharmacological data revealed the usefulness of binding methods in the further study of adrenergic mechanisms in the canine saphenous vein.     

Identification of α2-Adrenergic Receptors in Chicken Pineal Gland Using [3H]Rauwolscine

The norepinephrine-induced inhibition of avian pineal N-acetyltransferase activity appears to be mediated by alpha 2-adrenergic receptors. In this study, alpha 2-adrenergic receptors in the chicken pineal gland were directly identified by radioligand binding. Membrane preparations of pineal glands from chickens from 1 to 6 weeks of age were examined using [3H]rauwolscine, a selective alpha 2-adrenergic receptor antagonist, to characterize the binding sites. The results indicate no ontological change in either the affinity (KD) or density of receptor binding sites (Bmax) during the time span examined. The binding was saturable and of high affinity with a mean KD of 0.27 +/- 0.01 nM and a mean Bmax of 242 +/- 12 fmol/mg protein. Further characterization of these binding sites indicated that the alpha 2-adrenergic receptor is of the alpha 2A subtype, since prazosin and ARC-239 bound with low affinities and oxymetazoline bound with high affinity.     

Identification of alpha1-adrenergic receptors and their involvement in phosphoinositide hydrolysis in the frog heart

The aim of this study was to characterize alpha(1)-adrenergic receptors in frog heart and to examine their related signal transduction pathway. alpha(1)-Adrenergic binding sites were studied in purified heart membranes using the specific alpha(1)-adrenergic antagonist [(3)H]prazosin. Analysis of the binding data indicated one class of binding sites displaying a K(d) of 4.19 +/- 0.56 nM and a B(max) of 14.66 +/- 1.61 fmol/mg original wet weight. Adrenaline, noradrenaline, or phenylephrine, in the presence of propranolol, competed with [(3)H]prazosin binding with a similar potency and a K(i) value of about 10 microM. The kinetics of adrenaline binding was closely related to its biological effect. Adrenaline concentration dependently increased the production of inositol phosphates in the heart in the presence or absence of propranolol. Maximal stimulation was about 8.5-fold, and the half-maximum effective concentration was 30 and 21 microM in the absence and presence of propranolol, respectively. These data clearly show that alpha(1)-adrenergic receptors are coupled to the phosphoinositide hydrolysis in frog heart. To our knowledge, this is the first direct evidence supporting the presence of functional alpha(1)-adrenergic receptors in the frog heart.     

Characterization of L-Glutamate Binding Sites in Rat Spinal Cord Synaptic Membranes: Evidence for Multiple Chloride Ion-Dependent Sites

The effects of various ions on L-glutamate (L-Glu) binding sites (Na+-dependent, Cl(-)-dependent, and Cl(-)-independent) in synaptic plasma membranes (SPM) isolated from rat spinal cord and forebrain were examined. Cl(-)-dependent binding sites were over twofold higher in spinal cord (Bmax = 152 +/- 34 pmol/mg protein) as compared to forebrain SPM (Bmax = 64 +/- 12 pmol/mg protein). Na+-dependent binding, on the other hand, was nearly sixfold less in spinal cord (Bmax = 74 +/- 10 pmol/mg protein) compared to forebrain SPM (408 +/- 26 pmol/mg protein). Uptake of L-Glu (Na+-dependent) was also eightfold less in the P2 fraction from spinal cord relative to forebrain (Vmax of 2.89 and 22.3 pmol/mg protein/min, respectively). The effects of Na+, K+, NH4+, and Ca2+ on L-Glu binding sites were similar in both regions of the CNS. In addition, in spinal cord membranes, Br-, I-, and NO3- were equivalent to Cl- in their capacity to stimulate L-Glu binding, whereas F- and CO3- were less effective. Cl(-)-dependent L-Glu binding in spinal cord membranes consisted of two distinct sites. The predominant site (74% of the total) had characteristics similar to the Cl(-)-dependent binding site in forebrain membranes [i.e., Ki values of 5.7 +/- 1.4 microM and 119 +/- 38 nM for 2-amino-4-phosphonobutyric acid (AP4) and quisqualic acid, (QUIS), respectively]. The other Cl(-)-dependent site was unaffected by AP4 but was blocked by QUIS (Ki = 14.2 +/- 4.8 microM).