Introduction

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₁-Adrenergic receptors (₁-AR) are cell-surface, heptahelical receptors of the G protein-coupled receptor superfamily that bind the endogenous catecholamines epinephrine and norepinephrine and mediate the actions of the sympathetic nervous system (Graham et al., 1995). Three ₁-AR subtypes, _1a-AR, _1b-AR, and _1d-AR, have been characterized from the cloning of their individual cDNAs (Lomasney et al., 1991; Perez et al., 1991; Cotecchia et al., 1988; Perez et al., 1994). Each of these subtypes signals by coupling to membrane-bound G proteins. The predominant second messenger pathway stimulated by ₁-ARs is the activation of phospholipase C, a membrane-bound enzyme that generates the formation of soluble inositol triphosphate (IP₃) and diacylglycerol from phosphatidylinositol-4,5-bisphosphate (Hwa et al., 1996). IP₃ and diacylglycerol subsequently cause the release of calcium from intracellular stores and stimulate protein kinase C, respectively (Berridge, 1993). ₁-ARs are distributed extensively throughout the tissues of the cardiovascular system, regulating the flow of blood through large conduit vessels and controlling the peripheral vascular resistance at the arteriolar level, the latter being an important determinant of the systemic blood pressure (Graham et al., 1995; Piascik et al., 1995; Hwa et al., 1996). ₁-ARs also are located on cardiomyocytes; current evidence suggests that the ₁-AR subtypes may play key and distinct roles in modulating the force and rate of cardiac contraction, especially during pathology. Specifically, the _1a-AR has been implicated in promoting abnormal heart rhythms in ischemia, whereas the activation of _1b-ARs is thought to promote normal heart rhythms (Anyukhovsky and Rosen, 1991; Anyukhovsky et al., 1992). Therefore, the activation of ₁-ARs by circulating epinephrine or norepinephrine released from sympathetic nerves must be carefully controlled to maintain cardiovascular homeostasis.

Benzodiazepines are widely used in clinical practice as a premedicant in surgery or a sedative-amnesic. After i.v. administration, benzodiazepines are rapidly distributed to the brain. Their principal molecular target is the -aminobutyric acid (GABA)_A receptor, a pentameric integral membrane ion channel. By themselves, benzodiazepines do not activate the GABA_A receptor, but instead act as allosteric modulators increasing the affinity and efficacy of the endogenous ligand, GABA, to bind and activate the receptor (Costa et al., 1975; Haefely et al., 1975). Activation of the GABA_A receptor causes a chloride ion influx that hyperpolarizes the cell, facilitating the inhibitory or sedative actions of benzodiazepines in the central nervous system. Additional studies have suggested that the anxiolytic actions of benzodiazepines use other independent molecular pathways distinct from those by which the sedative actions of benzodiazepines are manifested. Indeed, the triazolobenzodiazepine alprazolam activates brain ₂-ARs in reserpine-treated rats and antagonizes the anxiogenic effects of yohimbine at these receptors (Eriksson et al., 1986). Other clinical studies to assess the effects of alprazolam on brain noradrenergic function have suggested that the antipanic mechanism of alprazolam may be due to an interaction between benzodiazepine-sensitive and noradrenergic neural systems (Charney and Heninger, 1985).

Significant hemodynamic alterations have been observed in vivo following the adminstration of benzodiazepines (Kotrly et al., 1984; Marty et al., 1986; Taneyama et al., 1993). These effects include decreases in the systemic blood pressure and variations ranging from mild decreases to modest increases in heart rate. These effects are mediated in part by the inhibitory actions of benzodiazepines on the sympathetic nervous system. In recent studies, we have demonstrated that individual benzodiazepines differentially inhibit intracellular calcium oscillations generated in response to the selective ₁-AR agonist phenylephrine in individual pulmonary artery smooth muscle cells (Hong et al., 1998). Although the addition of lorazepam produced a concentration-dependent decrease in the amplitude of the calcium oscillation in these cells, diazepam resulted in concentration-dependent decreases in the frequency of the oscillations. These inhibitory actions on calcium oscillations in an arterial smooth muscle cell in vitro may conceivably explain the in vivo observations of blood vessel dilation following benzodiazepine administration (Chang et al., 1994). In the present study, we have performed experiments to test our hypothesis that benzodiazepines modulate the signaling responses of ₁-AR agonists via a direct interaction with the ₁-AR. We have used radioligand binding experiments to determine the affinities with which benzodiazepines occupy binding sites on each of the ₁-AR subtypes. In addition, we have measured total inositol phosphate (IP) levels in rat fibroblasts expressing single ₁-AR populations to characterize the inherent signaling properties of benzodiazepines and to assess their ability to modify the signaling properties of agonists at each of the ₁-AR subtypes. In summary, we report our findings of a novel sympathomimetic property of benzodiazepines and the potential role of allosterism in modulation of ₁-AR function.

	Experimental Procedures

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Materials. Drugs were obtained from the following manufacturers: ()-epinephrine, phenylephrine, and geneticin, Sigma Chemical Co., St. Louis, MO; [¹²⁵I]2-[-(4-hydroxyl-3-[¹²⁵I]iodophenyl)ethylamineomethyl]tetralone ([¹²⁵I]HEAT), [³H]myo-inositol, [³H]muscimol, and [³H]flunitrazepam, DuPont NEN, Boston, MA; PK11195 and clonidine, Research Biochemicals Inc., Natick, MA; lorazepam (Ativan), Wyeth Laboratories, Andover, PA; midazolam (Versed), Roche Laboratories, Nutley, NJ; and diazepam (Elkins-Sinn, Inc., Cherry Hill, NJ).

Cell Culture and Transfection. COS-1 cells (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin and streptomycin. Cells were maintained and passaged upon reaching confluency by standard cell culture techniques. Experiments were conducted on cells between passages 10 and 25. Cells were transiently transfected with the DEAE-dextran method previously described with the cDNA of a single subtype of ₁-AR, subcloned into the eukaryotic expression plasmid pMT2' (Perez et al., 1991). Stably transfected rat-1 fibroblasts that express a single human ₁-AR subtype were maintained in continuous culture in DMEM supplemented with 10% (v/v) FBS and 500 µg/ml geneticin. The expression level of receptors on the rat-1 fibroblasts ranged from 5.5 to 9 pmol/mg membrane protein.

Membrane Preparation. Transiently transfected COS-1 cells were scraped 72 h post-transfection, collected, and washed in Hanks' balanced salt solution, then pelleted under low-speed centrifugation (1260g for 5 min). The cell pellet was resuspended in a 0.25 M sucrose solution and after another low-speed centrifugation step, the pellet was resuspended in a 10-ml volume of water containing a cocktail of protease inhibitors (40 µg of leupeptin,68 µg of phenylmethylsulfonyl fluoride, 400 µg of bacitracin, and 400 µg of benzamidine) and frozen at 70°C for 30 min. Membranes were prepared from the cell suspension by 20 strokes of a "B" glass Dounce homogenizer. Nuclear debris was removed by a low-speed centrifugation step. Membranes in the supernatant were washed with HEM buffer [20 mM HEPES, pH 7.4, 1.4 mM ethylene glycol bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 12.5 mM MgCl₂] and pelleted by high-speed centrifugation (30,000g for 15 min). Two additional washes of the membrane pellet with HEM buffer (20 ml) were performed, and the final pellet was reconstituted in a known volume of HEM buffer containing 10% (v/v) glycerol and stored at 70°C until use. The protein concentration of the membrane preparation was determined by performing a Bradford assay (Bradford, 1976), with bovine serum albumin as the known standard.

Measurement of Ligand-Binding Affinities. The binding affinities of benzodiazepines were determined in a series of competition-binding experiments with the ₁-AR-selective antagonist [¹²⁵I]HEAT as the radioligand. Assays were performed in duplicate in HEM buffer, in a total assay volume of 250 µl. A fixed amount of COS-1 cell membranes expressing a single ₁-AR subtype was incubated with 100 pM [¹²⁵I]HEAT and a range of 10 different concentrations of the competing benzodiazepine. Nonspecific binding was determined experimentally in the presence of 10⁴ M phentolamine. After incubation in a shaking water bath at 22°C for 60 min, unbound radioactivity was separated from membrane-bound radioactivity by filtration through Whatman GF/C filter paper with a Brandel cell harvester (Brandel, Gaithersburg, MD). Filters were washed with 20 ml of ice-cold HEM buffer to remove further nonspecifically bound radioactivity. Bound radioactivity remaining on the filters was counted on an ICN gamma counter operating at 79.8% efficiency.

Quantitation of Intracellular IP. Rat-1 fibroblasts plated on 60-mm culture plates were grown in DMEM supplemented with 5% FBS. Upon reaching 90% confluency, 3 µC_i of [³H]myo-inositol was added 16 h before experimentation to permit uptake by the fibroblasts. Measurement of intracellular IP was performed under serum-free conditions by washing fibroblasts with 10 ml of serum-free DMEM. To prevent complete hydrolysis of IP moieties, assays were conducted in the presence of the phosphatase inhibitor LiCl (10 mM) in a total assay volume of 3 ml. Agonists were added directly to the media and incubated at 37°C for 45 min in a 5% CO₂ atmosphere. In certain studies, antagonists were added 30 min before the addition of the agonist. Complete concentration-response curves for agonists were constructed over a suitable range of concentration, with at least two concentrations per order of magnitude and performing each data point in duplicate. Incubations were terminated by removal of the media containing the agonist and by adding a 1-ml volume of a 0.4 M perchloric acid solution. The cell lysate was scraped, collected, and neutralized by the addition of a 0.5-ml volume of a 0.72 N KOH/0.6 M KHCO₃ solution. Soluble IPs in the lysate were isolated by passage through a Bio-Rad AG 1X-8 resin column that was buffered with a 0.1 M formic acid solution. After washing the column with 0.1 M formic acid, bound ³H-IPs were displaced from the column by eluting the column with a 0.1 M formic acid solution containing 1 M ammonium formate. The eluant was collected directly in scintillation vials, scintillant was added, and the radioactivity was detected with a beta-counter (Beckman Instruments, Berkeley, CA).

Data Analysis. Competition-binding data and functional data from intracellular IP measurements were analyzed with the nonlinear regression functions of the noniterative curve-fitting program GraphPad Prism. Binding affinities (K_i) were determined by transformation of the program-calculated IC₅₀ value with the Cheng-Prusoff equation. The binding data for benzodiazepine binding was modeled to one- or two-site binding. The most suitable model was determined by performing an F test comparison of the least sum-of squares fit of the data to these equations. Functional data for IP stimulation in fibroblasts were analyzed by nonlinear regression analysis, with the sigmoidal curve-fitting equation of GraphPad Prism. In these studies, potency refers to the concentration of the ₁-AR agonist that stimulates the half-maximal IP response and was calculated directly from nonlinear regression analysis. Statistically significant differences in the potency of responses relative to control were determined by t test analysis. Statistically significant differences in the maximal responses from control were determined with a repeated measures two-tailed analysis of variance test followed by a post hoc Dunnett's multiple comparison test.

	Results

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Binding of Benzodiazepines to ₁-ARs Binding studies were performed on COS-1 cell membranes that had been transiently transfected to singly express one of the three ₁-AR subtypes. The affinities of diazepam, lorazepam, and midazolam at each of the ₁-AR subtypes were determined in heterologous competition-binding assays, measuring the ability of each of these drugs to compete for binding sites on the receptors that were labeled with the selective ₁-AR antagonist [¹²⁵I]HEAT. Increasing concentrations of diazepam, lorazepam, and midazolam resulted in concomitant decreases in the specific binding of [¹²⁵I]HEAT at each of the ₁-AR subtypes. Complete displacement of [¹²⁵I]HEAT binding was observed with each of these benzodiazepines at each ₁-AR subtype. Analysis of the data with nonlinear regression indicated that a single-site model was the most appropriate fit of the inhibition curves generated for benzodiazepine binding at each of the ₁-ARs. A composite displacement curve for midazolam is shown in Fig. 1 and the affinities of individual benzodiazepines at the ₁-AR subtypes are listed in Table 1.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Binding of midazolam to 1-AR subtypes. Inhibition of specific [125I]HEAT binding by midazolam at 1a-AR (), 1b-AR (), and 1d-AR (). Data points on the curves represent the mean ± S.E. value calculated from at least three individual experiments.

View this table: [in this window] [in a new window]   TABLE 1 Binding affinities of benzodiazepines at 1-ARs Binding affinities (Ki) were determined from competition binding studies displacing the 1-AR selective radioligand [125I]HEAT from 1-AR subtypes expressed individually on COS-1 cell membranes.

Effects of Benzodiazepines on Signaling Properties of ₁-ARs. Because these benzodiazepines demonstrated the ability to occupy binding sites on the ₁-AR subtypes, additional experiments were conducted to examine the signaling properties of benzodiazepines at these receptors. Because the predominant signaling pathway for ₁-ARs is activation of phospholipase C via G_q coupling, we measured the intracellular levels of IPs in rat-1 fibroblasts that are stably transfected to express a single ₁-AR subtype. These cell lines have two distinct advantages over the use of transient transfection of COS-1 cells in signaling studies. These fibroblasts maintain a uniform and high level of ₁-AR expression, limiting the variability between experiments that could result from variations in transfection efficiency. In addition, the stimulation resulting from the challenge with agonists produces large increases over the basal levels measured in the absence of agonist, thereby enhancing the ability to detect small changes in either the potency (EC₅₀) or maximal responses. Indeed, a full agonist at the _1a-AR in these rat-1 fibroblasts can produce specific increases in IP release up to 40,000 cpm (~100-fold). Experiments were conducted to observe the effects of benzodiazepines on ₁-AR-mediated IP accumulation in the presence and absence of ₁-AR agonists.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Benzodiazepines possess weak intrinsic activity in stimulating IPs via their interaction with 1-ARs. A, concentration-dependent stimulation of IPs by each of diazepam (DIA), lorazepam (LOR), and midazolam (MID) in rat-1 fibroblasts expressing the 1a-AR. Number shown represents the log of the molar concentration of drug added. The phenylephrine (PHE) response at a concentration of 103 M is shown for comparison. B, IP responses to 1 mM diazepam (DIA), lorazepam (LOR), or midazolam (MID) in rat-1 fibroblasts expressing the human 1a-AR are reversed by the addition of the 1-AR antagonist prazosin (1 µM; praz) but not by a similar saturating concentration (1 µM) of the GABA receptor antagonist PK11195 (PK). Data are shown as the means ± S.E. of four experiments, each concentration being performed in duplicate in each individual assay.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Synergistic interaction of 1-AR agonists and benzodiazepines in stimulating intracellular IP levels. Stimulation of IPs by increasing concentrations of phenylephrine in the absence (; thick line) or presence of 100 µM diazepam (), lorazepam (), or midazolam () in rat-1 fibroblasts expressing the human 1a-AR (A), human 1b-AR (B), and human 1d-AR (C). Each data point is the mean of three individual experiments in which each data point is performed in duplicate.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Modulation of phenylephrine responses is due to benzodiazepine interactions at 1-ARs. Generation of phenylephrine-induced increases in IPs in the absence (; thick line) or presence of 100 µM midazolam (), 100 µM midazolam, and 1 µM prazosin () or 100 µM midazolam and 1 µM PK11195 () in rat-1 fibroblasts expressing the human 1b-AR. Data points are shown as the mean ± S.E. generated from three individual experiments performed in duplicate.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Synergistic potentiation of a partial agonist response at the 1a-AR in the presence of benzodiazepines. IP response to increasing concentrations of clonidine in the absence (; thick line) and presence of 100 µM diazepam (), 100 µM lorazepam (), or 100 µM midazolam () in rat-1 fibroblasts expressing the 1a-AR. Data are shown as means ± S.E. from three individual experiments performed in duplicate.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Synergistic potentiation of a full agonist response by benzodiazepines at the 1b-AR. Generation of epinephrine-induced increases in IPs in the absence (; thick line) or presence of 100 µM diazepam (), 100 µM lorazepam (), or 100 µM midazolam () in rat-1 fibroblasts expressing the 1b-AR. Data are shown as means ± S.E. from three individual experiments performed in duplicate.

Mechanism of Diazepam Inhibition at _1a-AR. In our previous experiments at the _1a-AR, a single concentration of diazepam inhibited the potency of phenylephrine without reducing the maximal response (Fig. 3A). To determine whether diazepam inhibits _1a-AR signaling via a competitive or noncompetitive interaction, experiments were conducted to observe how increasing concentrations of diazepam would effect the signaling response to phenylephrine. As shown in Fig. 7, increasing concentrations of diazepam did not produce parallel rightwards shifts of the phenylephrine concentration-response curve at the _1a-AR that are indicative of a competitive interaction. Instead, the inhibitory profile illustrates an effect consistent with that of a noncompetitive interaction between diazepam and phenylephrine.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   The synergistic action of benzodiazepines in modulating 1-AR function is due to a noncompetitive mechanism. Phenylephrine-induced increases in IPs in the absence (; thick line) or presence of 100 µM diazepam (), 300 µM diazepam, (), or 1000 µM diazepam () in rat-1 fibroblasts expressing the 1a-AR. Data are shown as means ± S.E. from three individual experiments performed in duplicate.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

Intravenous anesthetics can differentially modulate the intracellular calcium oscillations in pulmonary artery smooth muscle cells stimulated by the ₁-AR-selective agonist phenylephrine (Hong et al., 1998). ₁-AR evoked intracellular calcium oscillations arise following phospholipase C activation that generates IP₃, which promotes calcium release from the sarcoplasmic reticulum (Berridge, 1993; Hwa et al., 1996). Because benzodiazepines are lipophilic, they may cross the plasma membrane and interact with numerous intracellular molecular targets along this signaling pathway. Alternatively, the benzodiazepines may alter the interaction of phenylephrine at the agonist binding pocket of the ₁-AR itself or modulate the ₁-AR function via the signaling cascade of the GABA receptor. Benzodiazepines, like ₁-AR ligands, possess a protonated amine functionality and retain a high degree of aromatic character that is found in many ₁-AR agonists and antagonists. Therefore, we rationalized that the similarity of their size and the conservation of these key sympathomimetic pharmacophores made an interaction at the ₁-AR the most likely site of action for these benzodiazepines. Accordingly, our experiments were conducted to investigate such an interaction of benzodiazepines with the ₁-AR, quantitating their affinity for the receptor and their effects on the signaling properties of the ₁-AR.

All three benzodiazepines used in our study inhibited the binding of the selective ₁-AR antagonist [¹²⁵I]HEAT from each of the ₁-AR subtypes. The affinities of the benzodiazepine at the ₁-ARs are listed together with our previously determined binding affinities of the full agonist epinephrine and the partial agonist methoxamine at ₁-ARs in Table 1. Although the benzodiazepine affinities are considerably lower than the affinity of epinephrine at all three subtypes, the affinities of diazepam and midazolam are only 2-fold lower than that of methoxamine at the _1a-AR and are actually 4-fold higher at the _1b-AR. Therefore, these benzodiazepines bind to and occupy sites on each of the three ₁-AR subtypes and with affinities that are comparable to other sympathomimetic drugs. Although this experimental method is designed to measure the competition between a drug and a radiolabel for a similar site on the receptor, this assay does not provide definitive proof of a competitive interaction. For example, allosteric modulators of muscarinic receptors can displace specific radioligand binding from these receptors in similar assays (Tucek and Proska, 1995). The determination of whether the interaction between benzodiazepines and [¹²⁵I]HEAT at ₁-ARs is a truly competitive or an allosteric interaction can only be determined from further detailed kinetic studies.

In signaling studies, each of the benzodiazepines demonstrated a concentration-dependent stimulation of IPs over basal levels in fibroblast cells expressing either the _1a-AR (Fig. 2A) or the _1b-AR (data not shown). Because these benzodiazepine-mediated increases in IPs could be reversed by the addition of prazosin, an ₁-AR antagonist, but not by PK11195, we conclude that the IP stimulation at the _1a-AR is due to an interaction of these benzodiazepines with the ₁-ARs and not with GABA receptors on the rat-1 fibroblast. Subsequent saturation-binding experiments with the GABA receptor ligands [³H]flunitrazepam or competitive-binding studies with [³H]muscimol failed to detect the presence of any GABA_A receptors on these fibroblasts (data not shown), confirming our conclusion that the weak partial agonist activity is due to their interaction with the ₁-AR.

The effects of the benzodiazepines on the signaling properties of the ₁-AR-selective agonist phenylephrine were subtype-dependent. At the _1a-AR, diazepam inhibited the potency of phenylephrine in stimulating IP; however, no statistically significant inhibition of the phenylephrine response was observed with midazolam or lorazepam. In fibroblasts expressing either the _1b-AR or _1d-AR subtype, the maximal response to phenylephrine in the presence of lorazepam and midazolam was markedly enhanced. We interpreted the differential benzodiazepine-mediated signaling inhibition or potentiation as being due to the intrinsic activity of phenylephrine, which is lower at the _1b-AR and _1d-AR subtypes compared with its full agonism at the _1a-AR. We confirmed our hypothesis by observing significant potentiation of the IP response to clonidine, a weak partial agonist at the _1a-AR subtype, in the presence of lorazepam and midazolam (Fig. 5). Therefore, we hypothesize that the intrinsic activity of the ₁-AR agonist is one determinant of whether potentiation of the signal is observed.

However, contrary to our expectations, both lorazepam and midazolam significantly increased the IP levels measured in response to the full agonists epinephrine (Fig. 6) and norepinephrine (data not shown) at the _1b-AR. The maximal stimulation by a full agonist at the _1a-AR produces levels of [³H]IP exceeding 40,000 cpm; however, under similar conditions, the full agonist epinephrine produced maximal responses of only 9,000 cpm at the _1b-AR. Because the receptors are expressed at similar receptor densities on the fibroblasts, it can be argued that the relative differences in [³H]IP levels reflect the greater signaling efficiency of the _1a-AR over the _1b-AR. Indeed, previous studies that titrate receptor density have shown that the _1a-AR subtype is always more efficacious than the _1b-AR or _1d-AR at any receptor number (Esbenshade et al., 1993; Theroux et al., 1996). It is conceivable that the binding of the benzodiazepine at the _1b-AR potentiates the response of the full agonist epinephrine by altering the receptor conformation to one that enhances the interaction of the receptor with the G protein, consistent with an allosteric effect. Therefore, our experiments have illustrated that the potentiating effects of benzodiazepines on ₁-AR signaling are not only dependent on the intrinsic activity of the agonist but also may be dependent on the efficacy with which the receptor is coupled to the signaling pathway.

The inhibitory profile of diazepam on phenylephrine-induced stimulation of IP at the ₁-AR illustrates an effect consistent with that of a noncompetitive interaction between phenylephrine and diazepam at this receptor (Fig. 7). The unequivocal demonstration of a noncompetitive interaction in these signaling studies is more definitive of an allosteric effect than by using the simple displacement of radiolabel binding at the receptor to determine the mechanism of the interaction. In addition, our other signaling studies demonstrated that lorazepam and midazolam potentiate the responses of full and partial agonists at the _1b-AR (Figs. 3B and Fig. 6). We argue that to observe signal potentiation of agonists requires the simultaneous occupation of the receptor by the benzodiazepine and the agonist, and thus separate binding sites exist for each compound on the receptor. Our observations allow us to speculate that the benzodiazepine is behaving as an allosteric modulator of ₁-AR function, in a similar fashion to their documented allosteric modulation of GABA binding and channel activation at the GABA_A receptor.

Recent studies have shown that aromatic and hydrophobic residues on the 1 and 2 subunits of the GABA_A receptor mediate benzodiazepine binding at the allosteric binding sites on these receptors (Wieland et al., 1992; Sigel and Buhr, 1997; Sigel et al., 1998). Likewise, the juxtamembrane regions of the transmembrane helices and the extracellular loops of the ₁-ARs contain numerous aromatic and hydrophobic amino acids that may facilitate binding of the benzodiazepines to the ₁-AR. Therefore, we speculate that the benzodiazepine binding site on ₁-ARs lies above the catecholamine binding pocket, previously mapped in the hydrophilic core within the circular array of transmembrane spanning helices of the _1b-AR (Hwa et al., 1995; Hwa and Perez, 1996). Alternatively, the lipophilic characteristics of benzodiazepines may facilitate their interaction with the lipophilic core of the ₁-AR, or the benzodiazepine may bind in a pocket formed within the circular array of transmembrane helices but on the opposite side from that of the agonists.

Our laboratory has proposed that the activation of ₁-ARs involves the agonist-mediated disruption of an interhelical salt bridge formed between an aspartic acid (Asp¹²⁵) in transmembrane 3 and a lysine residue (Lys³³¹) in transmembrane 7 (Porter et al., 1996, 1998). Our model predicts that the protonated amine group of the agonist projects toward and forms an ion-pair with Asp¹²⁵ after salt bridge breakage. We speculate that the protonated amine group present on the benzodiazepine is orientated so that it too can project toward Asp¹²⁵ and break the salt bridge even when the agonist binding pocket is occupied. When the ₁-AR is occupied by a partial agonist, the simultaneous interaction of the benzodiazepine's protonated amine provides additional energy to release the interhelical salt-bridge conformational restraint and potentiates the actions of the agonist. This is consistent with our experimental observations in which the greatest signal potentiation occurs when a weak partial agonist occupies the agonist binding pocket of the ₁-AR, e.g., clonidine at the _1a-AR (Fig. 5). In contrast, a full agonist possesses sufficient intrinsic strength to disrupt the constraining salt bridge itself and requires no additional energetic requirement from the benzodiazepine to activate the receptor, e.g., phenylephrine at the _1a-AR (Fig. 3A). Indeed, at high concentrations (1 mM), benzodiazepines actually inhibit the signal of the full agonist (Fig. 7), an effect probably the result of steric hindrance. Because receptor efficacy also determines benzodiazepine potentiation, we predict that the alignment and orientation of epinephrine in the binding pockets of the _1a-AR and _1b-AR are slightly different. A closer projection of the protonated amine group of epinephrine toward the aspartic acid residue in the _1a-AR would be more energetically favorable for salt bridge breakage and explain the minimal effects of the benzodiazepine at the _1a-AR. This hypothesis is based upon our previously reported observations with triethylamine, a chemical mimic of the ethylamine substituent in epinephrine, to also potentiate clonidine but not epinephrine signaling at the _1a-AR (Porter et al., 1998). Indeed it is important to note that the protonated amine group in triethylamine and benzodiazepines is the only commonality between the two potentiators. If our hypothesis is correct, the degree of potentiation depends upon the efficacy of the system, whether it is controlled at the level of the agonist (i.e., greatest potentiation with the weakest agonist) or at the level of the receptor (i.e., greatest potentiation with the weakest coupling).

In summary, we have reported the findings that three benzodiazepines, diazepam, lorazepam, and midazolam, bind to and modulate the intracellular signaling of all three ₁-AR subtypes, probably through an allosteric interaction at the receptor level. Benzodiazepines possess low intrinsic activity in stimulating IPs but exert profound effects on the signaling properties of full and partial agonists at ₁-ARs. Although our observations are made at concentrations 100-fold in excess of the reported 1 µM plasma concentration of benzodiazepines (Gamble et al., 1976; Dundee et al., 1978), the blood chemistry of lipophilic agents makes accurate determination of their concentration difficult, and the possibility remains that the blood levels may be higher than reported. However, it is unlikely that the clinical administration of benzodiazepines will result in their cross-reactivity with adrenergic receptors. Nevertheless, the actions of benzodiazepines at ₁-ARs may represent part of a general allosteric site on the ₁-AR, similar to the binding of amiloride analogs at the ₂-AR (Leppik et al., 1998) and suggest that other higher-affinity modulators of adrenergic receptors may exist. Such a site to modulate ₁-AR function may be therapeutically useful such as with the allosteric modulators at the muscarinic acetylcholine receptors (Tucek and Proska, 1995) and GABA_A receptors (Sigel and Buhr, 1997).