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Previous studies have shown that neural stimulation of brown adipose tissue (BAT) reorganizes the expression and activity of signaling proteins in the beta-adrenergic adenylyl cyclase pathway. Cold stress increases neural stimulation of BAT and increases alpha1-adrenergic receptor number; however, the alpha1 receptor subtype involved and the mechanism of up-regulation by cold stress have not been determined. Using reverse transcription/polymerase chain reaction analysis and nuclease protection assay, BAT was demonstrated to express mRNAs encoding alpha1a and alpha1d, but not alpha1b, receptors. Parallel pharmacologic studies of BAT membranes and recombinant alpha1a and alpha1d receptors expressed in COS-7 cells demonstrated that alpha1a receptors predominate in BAT. Exposure of rats to 4 degrees for 4 days increased alpha1a receptors and mRNA in BAT but did not alter expression of alpha1d receptors or mRNA. The induction of alpha1a receptor and mRNA level by cold stress was prevented by selective surgical denervation of BAT. Furthermore, alpha1a receptor and mRNA expression could be induced in warm-adapted rats by infusions of the selective beta3-adrenergic receptor agonist CL 316,243. These data indicate that neural activation of beta3-adrenergic receptors is an important determinant of alpha1a adrenergic receptor expression in BAT.

A cysteine-to-phenylalanine mutation in the third transmembrane domain of the alpha 1B-adrenergic receptor constitutively activates the receptor, resulting in G protein coupling in the absence of agonist and activation of only a single effector pathway (phospholipase C but not phospholipase A2). This mutant receptor displays a higher affinity for the catecholamines, norepinephrine, and epinephrine, as well as for other phenethylamines, but not for imidazolines, a class of structurally distinct alpha agonists. Dose-response studies demonstrate a higher potency and intrinsic activity of phenethylamines for polyphosphoinositide turnover but not for arachidonic acid release. Imidazolines have wild-type potencies and intrinsic activities for both pathways. These data indicate that a single receptor subtype forms multiple conformations (i.e., exhibits induced conformational pleiotropy) for G protein interactions (high affinity states) that are specific for a particular G protein/effector pathway and that multiple binding sites exist for agonists, which promote or induce these specific interactions. Pharmacological diversity may, thus, be achieved through a single receptor by the development of compounds that induce a single activated conformer. This has major ramifications for the eventual development of signaling-specific therapeutics.

We have cloned the human alpha 1d-adrenergic receptor (AR) and compared the pharmacological properties of the three recombinant human alpha 1-AR subtypes in SK-N-MC cells. SK-N-MC cells natively express a mixture of alpha 1-AR subtypes, and the use of an inducible expression system allowed us to directly compare the recombinant and native subtypes without concern for cell-specific processing or microenvironment. The human alpha 1d-AR was expressed from a cDNA/gene fusion construct cloned from human SK-N-MC cell cDNA and human genomic libraries. This receptor is deduced to contain 572 amino acids with 98% identity to the rat alpha 1d-AR in the transmembrane domains and, when expressed in human embryonic kidney 293 cells, has alpha 1-AR binding properties similar to those of the rat alpha 1d-AR. Norepinephrine increased inositol phosphate formation and mobilized intracellular Ca2+ in transfected 293 cells. Reverse transcription-polymerase chain reaction analysis of the three cloned human subtypes (alpha 1a, alpha 1b, and alpha 1d) in mRNA from SK-N-MC cells, which natively express alpha 1A- and alpha 1B-like pharmacology, showed abundant alpha 1a and alpha 1d but fewer alpha 1b transcripts. The three human clones were expressed in SK-N-MC cells using isopropyl-beta-D-thiogalactoside-inducible vectors. Upon induction, alpha 1-AR density was increased with the recombinant subtype comprising 67-80% of total alpha 1-ARs. Inhibition curves for (+)-niguldipine and 5-methylurapidil fit best to a two-site model in uninduced cells, indicating significant receptor heterogeneity. Isopropyl-beta-D-thiogalactoside induction altered the potencies of both compounds, causing most inhibition curves to fit best to a one-site model. (+)-Niguldipine was 100-fold more potent at the alpha 1a-AR than at alpha 1b- or alpha 1d-ARs, whereas 5-methylurapidil had similar potencies at alpha 1a- and alpha 1d-ARs and about 10-fold lower affinity at the alpha 1b-AR. We conclude that the complex alpha 1A- and alpha 1B-like pharmacology observed in native SK-N-MC cells is due to expression of all three subtypes in different proportions, independently of cell-specific processing or environmental factors, and that the alpha 1a-AR cDNA encodes the pharmacologically defined alpha 1A subtype.

The potencies and intrinsic activities of agonists in activating cloned alpha 1-adrenergic receptor (AR) subtypes were compared. The hamster alpha 1B-, bovine alpha 1C-, or rat alpha 1A/D-ARs were expressed at high levels in human embryonic kidney 293 cells. Catecholamines and phenylethylamines, but not lower efficacy agonists, were more potent in inhibiting radioligand binding to the expressed alpha 1A/D subtype than to the alpha 1B or alpha 1C subtypes; this selectivity remained in the presence of different buffers, nucleotides, and cations. Activation of all three subtypes caused substantial increases in [3H]inositol phosphate formation in cells grown in 96-well plates. Pretreatment with phenoxybenzamine decreased maximal responses to norepinephrine (NE) with only small decreases in apparent potency, suggesting similar small receptor reserves for all three subtypes. The catecholamines NE, epinephrine, and 6-fluoro-NE were full agonists with similar potencies at the three subtypes; alpha-methyl-NE was also a full agonist but was about 20-fold less potent at alpha 1B-ARs than at alpha 1C- or alpha 1A/D-ARs. Phenylephrine had similar potencies at all three subtypes but gave a submaximal response at alpha 1B-ARs. Methoxamine was a full agonist at alpha 1C- and alpha 1A/D-ARs, with about 20-fold greater potency at the alpha 1C subtype, but showed lower intrinsic activity at alpha 1B-ARs. A number of imidazolines, amidephrine, and SKF 89748 had substantial intrinsic activity at alpha 1C-ARs but little or no intrinsic activity at the other two subtypes. We conclude that the potencies of many agonists in competing for radioligand binding sites are related to their potencies in activating functional responses but that this relationship is not the same for all subtypes. NE and epinephrine activate all three cloned alpha 1-AR subtypes with similar potencies and intrinsic activities, but many widely used agonists show significant selectivity for different alpha 1-AR subtypes.

alpha 1-Adrenergic receptor (alpha 1-AR) agonists elevate the intracellular calcium concentration ([Ca2+]i) in 60-80% of astroglia in vitro. Likewise, 60-70% of astroglia exhibit specific binding sites for the alpha 1-AR-selective antagonist (+-)-125I-[beta-(4-hydroxyphenyl)-ethylaminomethyl]tetralone. The density of alpha 1-AR binding sites varies markedly on individual cells, ranging from a few to > 2000 binding sites/1000 microns 2 of surface area. In the present study, we examined the relationship between the density of alpha 1-AR binding sites on astroglia and their ability to respond to alpha 1-AR agonists with a rise in [Ca2+]i. A video-based imaging system was used to monitor calcium responses in individual astroglial cells, which were subsequently assessed for their expression of alpha 1-ARs using receptor binding autoradiography. The ability of a given concentration of phenylephrine (PE) to elicit a calcium response correlated well with alpha 1-AR density (r = 0.94), i.e., the higher the receptor density the greater the probability that a given astroglial cell would respond to alpha 1-AR agonists. However, the amplitude of the calcium response did not correlate with the alpha 1-AR density. Cells with low alpha 1-AR density (< 10 binding sites/1000 microns 2) could generate a response with an amplitude comparable to that seen in cells with high alpha 1-AR density (> 1000 binding sites/1000 microns 2). To evaluate the relationship between receptor occupancy and calcium response, PE concentrations and alpha 1-AR density were varied while the calcium response in individual cells was monitored. Interestingly, for a given cell the amplitude of calcium response reached its maximum with a small step increase in the concentration of PE (< 5-fold), whereas the latency of the response decreased when PE concentrations were increased. Irreversible inactivation of alpha 1-ARs by phenoxybenzamine reduced the potency of PE but not the maximal calcium response. Cells that responded to 100 nM PE were able to generate a comparable response to 10 microM PE after inactivation of 90% of the total alpha 1-AR binding sites with phenoxybenzamine treatment. In summary, our results indicate that most astroglial cells express a substantial level of "spare" alpha 1-ARs that increase the sensitivity of these cells to alpha 1-AR agonists. Once activated, individual astroglial cells tend to generate a maximal [Ca2+]i elevation that is independent of the total alpha 1-AR density or the concentration of ligand.

In isolated rat white adipocytes, epinephrine (in the presence of 10 microM propranolol) increased the uptake of [32P]Pi into phosphatidylinositol in a dose-dependent fashion. When the cells were pretreated with the irreversible antagonist chlorethylclonidine, this alpha 1-adrenergic effect was markedly diminished. The effect of epinephrine was dose-dependently antagonized by selective alpha 1-adrenergic antagonists, with the potency order prazosin greater than 5-methylurapidil greater than or equal to WB4101. Binding studies using crude membrane preparations were performed with the ligands [3H]bunazosin and 125I-HEAT. Both ligands bound to membrane sites with high affinity (Kd values of 0.75 +/- 0.20 nM for [3H]bunazosin and 125 +/- 20 pM for 125I-HEAT), in a rapid, reversible, and saturable (Bmax, 9-12 fmol/mg of protein) fashion, and with the expected pharmacological characteristics for alpha 1-adrenoceptors. Binding displacement studies with these ligands indicated a potency order of prazosin greater than 5-methylurapidil greater than or equal to WB4101. Northern blot analysis using receptor subtype-specific gene probes showed that adipocyte mRNA hybridized with the alpha 1B-adrenergic probe. All these data suggest that the alpha 1-adrenoceptors of rat white adipocytes belong to the alpha 1B subtype.

Using the alpha 1-adrenoceptor subtype-selective antagonists chlorethylclonidine (CEC), WB4101, and 5-methyl-urapidil, we have examined the possible heterogeneity in the alpha 1-adrenoceptor populations in rabbit aorta. The alpha 1-adrenoceptor alkylating agent CEC selectively inhibited the phasic component of the norepinephrine-induced contractile response, with little effect on the tonic component. The alpha 1-adrenoceptor occupancy-response relationship defined by the phenoxybenzamine inactivation method was rectangular hyperbolic for the tonic response, whereas that for the phasic response was linear, indicating the different degree of receptor reserve for the two responses. Radioligand binding studies with the nonselective alpha 1-adrenoceptor antagonist radioligand 125I-BE2254 showed that 73-87% of the binding sites in rabbit aorta are CEC sensitive and they are predominantly low affinity sites both for WB4101 (pKd = 8.1) and for 5-methylurapidil (pKd = 7.1). Moreover, alpha 1-adrenoceptor-mediated phosphatidylinositol (PI) hydrolysis was CEC sensitive, and fractional inactivation of alpha 1 receptors with CEC showed equivalent increments in the reduction of PI hydrolysis and phasic contractile response, suggesting that both responses are linearly related to the CEC-sensitive receptor sites. The Schild plots for the competitive antagonists WB4101 and 5-methyl-urapidil against alpha 1a-adrenoceptor-selective agonist methoxamine-induced contraction were linear and had slopes not significantly different from unity, with a pA2 of 9.07 +/- 0.07 (n = 5) for WB4101 and 9.09 +/- 0.05 (n = 3) for 5-methyl-urapidil. However, the Schilod plots for these antagonists against norepinephrine were curvilinear. Computer-assisted analysis of these curvilinear Schild plots in a two-receptor system indicated that alpha 1-adrenoceptor populations responsible for the constrictive response are predominantly (approximately 80-90%) low affinity sites for the two antagonists (pKd approximately 8.1 for WB4101 and pKd approximately 7.1 for 5-methyl-urapidil) and a small population (approximately 10-20%) are high affinity sites (pKd approximately 9.1 for both WB4101 and 5-methyl-urapidil), which was in good agreement with radioligand binding studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Norepinephrine (NE) increased formation of [3H]inositol phosphates ( [3H]InsPs) in primary cultures of neuronal and glial cells from 1-day-old rat brain. This response appeared to be mediated by alpha 1-adrenergic receptors, because prazosin was 40-fold more potent than yohimbine in blocking it. Pretreatment with pertussis toxin (PTX) dose-dependently decreased this response by 70-80%. The IC50 for PTX (7 ng/ml) was similar to that for blocking of alpha 2-adrenergic receptor-mediated decreases in cyclic AMP accumulation in the same cells. PTX pretreatment caused only a small, not statistically significant, inhibition of the [3H]InsP response to the muscarinic cholinergic receptor agonist carbachol in these cells. Radioligand binding studies showed that both neuronal and glial cultures contained mixed populations of alpha 1a- and alpha 1b-adrenergic receptor subtypes. Selective inactivation of the alpha 1b population by chloroethylclonidine reduced NE-stimulated [3H]InsP formation by 25 +/- 6%. Pretreatment with both PTX and chloroethylclonidine caused additive decreases (90 +/- 3%) in the NE response. NE-stimulated [3H]InsP formation was partially dependent on extracellular calcium, because it was decreased 64 +/- 6% by removal of calcium and 56 +/- 13% by addition of 1 mM CdCl2, although it was not affected by 1 microM nifedipine. These results suggest that NE stimulates [3H]InsP formation in neuronal and glial cultures through a pertussis toxin-sensitive guanine nucleotide-binding protein. This response appears to be mediated primarily by the alpha 1a subtype and may be subsequent to calcium influx.

The interaction of agonists and antagonists with alpha 1-adrenergic receptors in rat vas deferens was examined using radioligand binding assays and contractility measurements. 125I-Labeled BE 2254 (125IBE) was found to bind rapidly and reversibly to a single class of high-affinity binding sites in homogenates of rat vas deferens. The k1 for association was 3.8 X 10(7) 1/mole-sec, the k-1 for dissociation was 2.3 X 10(-3) sec-1, and the KD was 105 pM. The order of potency for antagonists inhibiting 125IBE binding was prazosin greater than indoramin greater than phentolamine greater than yohimbine. Norepinephrine, phenylephrine, and other alpha-adrenergic agonists produced dose-dependent contractions of whole vas deferens in vitro. This contractile response was competitively inhibited by alpha-adrenergic blocking drugs with the same potency order observed for inhibition of specific 125IBE binding. Comparison of pA2 values for alpha 1- and alpha 2-selective antagonists competitively inhibiting contractile responses to norepinephrine, epinephrine, or phenylephrine suggested that these drugs caused their contractile effects solely through alpha 1-adrenergic receptors, and that there were no alpha 2-adrenergic receptors mediating contraction in this tissue. The pA2 values for antagonist inhibition of alpha-adrenergic receptor-mediated contractile responses were highly correlated (r = 0.995) with the KD values for antagonist inhibition of 125IBE binding in this tissue. The EC50 values for partial agonists were also highly correlated with the KD values for inhibition of 125IBE binding in vas deferens. However, the EC50 values of full agonists in causing contraction were in general 10- to 100-fold lower than the KD values for inhibiting 125IBE binding, possibly representing a substantial "spare receptor" population in this tissue. The results suggest that rat vas deferens contains a homogeneous population of alpha 1-adrenergic receptors mediating the contractile response to norepinephrine, that these receptors can be directly labeled with 125IBE, and that there may be a nonlinear relationship between agonist occupancy of alpha 1-adrenergic receptors and the functional response of this tissue.

Chlorethylclonidine (CEC) has previously been shown to inactivate only a subpopulation of the alpha 1-adrenergic receptor binding sites in rat brain. We compared alpha 1-adrenergic receptors in different tissues to determine whether such selective inactivation might reveal the presence of distinct receptor subtypes. Pretreatment of broken cell preparations with 10 microM CEC for 10 min caused a 70-80% decrease in the density of specific 125IBE 2254 binding sites in rat liver and spleen, a 25% decrease in neocortex, but no significant loss in kidney, hippocampus, heart, vas deferens, or caudal artery. The effect of CEC in liver was not reversed by extensive washing, suggesting irreversible inactivation. The selectivity between different tissues was due to differences in the efficacy of CEC inactivating the binding sites and not due to differences in binding affinity. To determine whether the effects on 125IBE 2254 binding reflected selective inactivation of functional receptors, contractile responses of rat spleen and vas deferens were examined. Pretreatment of intact tissues with 100 microM CEC for 30 min caused a large decrease in the potency and maximal contraction to norepinephrine in spleen but had no effect in vas deferens. Inhibition of specific 125IBE 2254 binding by various agonists and antagonists was determined in CEC-sensitive (liver, spleen) and insensitive (hippocampus, vas deferens) tissues. Although many drugs had similar affinities in all tissues, others were substantially less potent in the CEC-sensitive tissues. These experiments suggest that there are at least two subtypes of alpha 1-adrenergic receptors with different pharmacological properties in mammalian tissues, only one of which is inactivated by CEC.

The interaction between some examples of mononuclear and binuclear DNA-intercalating antitumor agents and alpha- and beta-adrenoceptors has been studied using radioligand-binding assays. Competition for 125I-BE 2254, [3H]rauwolscine, and (-)-[3H]dihydroalprenolol binding was used to assess affinity for alpha 1-, alpha 2-, and beta-adrenoceptor-binding sites, respectively. Two homologous series of alkyl-linked diacridines and diquinolines were found to interact poorly with beta-adrenoceptors, with only the largest members having appreciable affinity. By contrast, these compounds bind strongly and in a complex manner to alpha 1- and alpha 2-adrenoceptors. The affinity of diacridines for both alpha-adrenoceptor classes has a parabolic dependence on alkyl chain length with the hexyl and pentyl derivatives being the most potent at the alpha 1- (Ki = 11.5 +/- 2.3 nM) and alpha 2- (Ki = 143 +/- 26 nM) binding sites, respectively. The dependence of inhibition constants on linker chain length for the diquinolines is more complicated, with the ethyl- and heptyl-linked dimers having the greatest affinity for each alpha subclass. There is a nadir in affinity for the pentyl and butyl ligands and an increase in dissociation constant for octyl and longer homologues. Thus, the ethyl diquinoline has Ki values of 6.6 +/- 1.2 and 110 +/- 14 nM for the alpha 1- and alpha 2- adrenoceptors, respectively, and, correspondingly, the heptyl derivative has values of 39 +/- 4 and 51 +/- 1 nM. These findings are discussed with respect to a model of the alpha-adrenoceptor in which the radioligand-binding site is situated in a trench or cleft, surrounded by a flat surface bounded by walls. Daunomycin was found to have no affinity for adrenoceptors of any type and mitoxantrone similarly fails to interact with alpha 2- and beta-adrenoceptors, but binds to the alpha 1 subclass with an inhibition constant (Ki) of 3930 +/- 420 nM. Bisantrene also has no affinity for beta-adrenoceptors but binds to alpha 1- and alpha 2- adrenoceptors with Ki values of 145 +/- 24 and 2310 +/- 430 nM, respectively. Among the mononuclear acridine drugs studied, only nitracrine shows detectable interaction with beta-adrenoceptors (Ki = 760 +/- 50 nM). This compound, like bisantrene, has high affinity for the alpha 1-adrenoceptor (Ki = 131 +/- 17 nM) and moderate affinity for the alpha 2 subclass (Ki = 2180 +/- 500 nM).(ABSTRACT TRUNCATED AT 400 WORDS)

We compared the effects of methoxamine on alpha 1-adrenergic receptor-mediated phosphorylase activation in rat hepatocytes and rabbit aorta. Although methoxamine is a potent agonist in activating phosphorylase of rabbit aorta, it had little effect in rat hepatocytes. Using the phenoxybenzamine inactivation method, we found that the quantitative relationship between 125I-BE2254 (125I-BE) binding capacity and maximal norepinephrine-stimulated phosphorylase activation was nonlinear in rabbit aorta, whereas it was linear in rat hepatocytes. The potency of methoxamine in inhibiting specific 125I-BE binding is significantly (p less than 0.05) higher in rabbit aorta (Kd, 96.4 +/- 7.7 microM), compared with rat hepatocytes (Kd, 283 +/- 16 microM). However, these quantitative differences could not fully explain the blunted [Ca2+]c and phosphorylase responses to methoxamine in rat hepatocytes. Treatment with chlorethylclonidine dose dependently suppressed 125I-BE binding sites and norepinephrine-induced phosphorylase activation in rat hepatocytes, whereas in rabbit aorta it resulted in only a 31% decrease in 125I-BE binding sites, with little effect on phosphorylase activation. Furthermore, alpha 1-adrenergic receptor-mediated cellular events of phosphatidylinositol (PI) hydrolysis and phosphorylase activation were unaffected by the removal of extracellular Ca2+ in rat hepatocytes, whereas both responses were markedly attenuated in rabbit aorta. The results indicate that two different alpha 1-adrenergic receptor subtypes activate glycogen phosphorylase, through different mechanisms for increasing [Ca2+]c in the two systems. In rat hepatocytes, alpha 1 receptors are closely linked to PI hydrolysis and Ca2+ release from intracellular stores and cause phosphorylase activation. In rabbit aorta, on the other hand, activation of alpha 1 receptors increases [Ca2+]c by Ca2+ influx from the extracellular fluid as well as by Ca2+ release, and both PI hydrolysis and phosphorylase activation are caused mainly by the Ca2+ entry. Methoxamine interacts with both chlorethylclonidine-sensitive and -insensitive alpha 1 receptor subtypes but selectively stimulates the alpha 1 receptor subtype that closely couples with the Ca2+ influx.

The existence of "spare" alpha 1-adrenergic receptors in rat vas deferens was examined directly using radioligand binding assays and contractility measurements. Alpha 1-adrenergic receptors in homogenates of rat vas deferens were labeled with [125I]BE 2254 (125IBE). Norepinephrine and other full alpha 1-adrenergic receptor agonists were much less potent in inhibiting 125IBE binding than in contracting the vas deferens in vitro. Treatment with 300 nM phenoxybenzamine for 10 min to irreversibly inactivate alpha 1-adrenergic receptors caused a large decrease in the potency of full agonists in causing contraction of this tissue and a 23-48% decrease in the maximal contraction observed. Using those data, equilibrium constants for activation (Kact values) of the receptors by agonists were calculated. These Kact values agreed well with the equilibrium binding constants (KD values) determined from displacement of 125IBE binding. The reduction in alpha 1-adrenergic receptor density following phenoxybenzamine treatment was determined by Scatchard analysis of specific 125IBE binding sites and compared with the expected reduction (q values) calculated from the agonist dose-response curves before and after phenoxybenzamine treatment. Exposure to 300 nM phenoxybenzamine for 10 min resulted in a 39% decrease in specific 125IBE binding sites, which did not agree with the 93% decrease expected from the calculated q values. Treatment of vas deferens with a dose of phenoxybenzamine (10 microM for 15 min) that completely abolished the contractile response to alpha 1-adrenergic agonists caused an 82% decrease in the density of 125IBE binding sites. Tissues exposed to 300 nM phenoxybenzamine in the presence of 100 microM phentolamine or 3 microM prazosin showed no change in the dose-response curves for agonist-induced contraction or in the density of 125IBE binding sites when compared with controls. This suggests that phenoxybenzamine functionally inactivates alpha 1-adrenergic receptors at or near the receptor binding site. These experiments suggest that the potencies of agonists in activating alpha 1-adrenergic receptors in rat vas deferens agree well with their potencies in binding to the receptors. The greater potency of agonists in causing contraction may be due to spare receptors in this tissue. The data also demonstrate that phenoxybenzamine irreversibly inactivates alpha 1-adrenergic receptors in rat vas deferens, but that the decrease in receptor density is much smaller than that predicted from receptor theory.

Activation of alpha 1-adrenergic receptors in slices of rat brain increases inositol phosphate accumulation, increases basal cyclic AMP accumulation, and potentiates the increase in cyclic AMP caused by adenosine. We compared these three responses to determine whether they are mediated by the same receptors. The increase in inositol phosphates and the potentiation of cyclic AMP accumulation in cerebral cortex were largely blocked by chelation of extracellular calcium, whereas the increase in basal cyclic AMP was not affected. The magnitude of the increase in inositol phosphates in different brain regions correlated with the magnitude of the potentiation of cyclic AMP accumulation (r = 0.80), but neither of these correlated with the magnitude of the increase in basal cyclic AMP. Although other alkylating agents inactivated all of the alpha 1-adrenergic receptor-binding sites labeled with 125IBE 2254 in membrane preparations of cerebral cortex, chlorethylclonidine (CEC) potently and selectively inactivated only half of these sites. Pretreatment with CEC partially blocked the increase in basal cyclic AMP, but not the increase in inositol phosphates or potentiation of cyclic AMP accumulation in slices of cerebral cortex. Comparing different brain regions, there was a better correlation between the density of 125IBE 2254-binding sites not inactivated by CEC with the magnitude of the increase in inositol phosphates or potentiation of cyclic AMP accumulation than with the increase in basal cyclic AMP. Although the largest increase in inositol phosphates was observed in slices of hippocampus, there was only a small increase in basal cyclic AMP in this region, and CEC did not inactivate any 125IBE-binding sites in hippocampus. Phentolamine and WB 4101 were significantly more potent in inhibiting specific 125IBE 2254 binding in hippocampus than in cerebral cortex. After treatment of cerebral cortical membranes with CEC, however, these drugs had potencies similar to those observed in hippocampus. The results suggest that the alpha 1-adrenergic receptors mediating increases in basal cyclic AMP accumulation can be differentiated from those mediating increases in inositol phosphate accumulation and potentiating adenosine stimulated cyclic AMP accumulation by their binding properties, calcium dependency, regional distribution, and sensitivity to the alkylating agent CEC.