Influence of the Membrane Dye R18 and of DMSO on Cell Penetration of Guanidinium-Rich Peptides.

A quantitative analysis by confocal fluorescence microscopy of the entry into HEK293 and MCF-7 cells by fluorescein-labeled octaarginine (1) and by three octa-Adp derivatives (2 - 4, octamers of the ß-Asp-Arg-dipeptide, derived from the biopolymer cyanophycin) is described, including the effects of the membrane dye R18 and of DMSO on cell penetration.

Synthesis, radiolabeling and evaluation of novel amine guanidine derivatives as potential positron emission tomography tracers for the ion channel of the N-methyl-d-aspartate receptor.

The N-Methyl-d-Aspartate receptor (NMDAR) is involved in many neurological and psychiatric disorders including Alzheimer's disease and schizophrenia. The aim of this study was to develop a positron emission tomography (PET) ligand to assess the bio-availability of the NMDAR ion channel in vivo. A series of tri-N-substituted diarylguanidines was synthesized and their in vitro binding affinities for the NMDAR ion channel assessed in rat forebrain membrane fractions. Compounds 21, 23 and 26 were radiolabeled with either carbon-11 or fluorine-18 and ex vivo biodistribution and metabolite studies were performed in Wistar rats. Biodistribution studies showed high uptake especially in prefrontal cortex and lowest uptake in cerebellum. Pre-treatment with MK-801, however, did not decrease uptake of the radiolabeled ligands. In addition, all three ligands showed fast metabolism.

Cell-Penetrating, Guanidinium-Rich Oligophosphoesters: Effective and Versatile Molecular Transporters for Drug and Probe Delivery.

The design, synthesis, and biological evaluation of a new family of highly effective cell-penetrating molecular transporters, guanidinium-rich oligophosphoesters, are described. These unique transporters are synthesized in two steps, irrespective of oligomer length, by the organocatalytic ring-opening polymerization (OROP) of 5-membered cyclic phospholane monomers followed by oligomer deprotection. Varying the initiating alcohol results in a wide variety of cargo attachment strategies for releasable or nonreleasable transporter applications. Initiation of oligomerization with a fluorescent probe produces, upon deprotection, a transporter-probe conjugate that is shown to readily enter multiple cell lines in a dose-dependent manner. These new transporters are superior in cell uptake to previously studied guanidinium-rich oligocarbonates and oligoarginines, showing over 2-fold higher uptake than the former and 6-fold higher uptake than the latter. Initiation with a protected thiol gives, upon deprotection, thiol-terminated transporters which can be thiol-click conjugated to a variety of probes, drugs and other cargos as exemplified by the conjugation and delivery of the model probe fluorescein-maleimide and the medicinal agent paclitaxel (PTX) into cells. Of particular significance given that drug resistance is a major cause of chemotherapy failure, the PTX-transporter conjugate, designed to evade Pgp export and release free PTX after cell entry, shows efficacy against PTX-resistant ovarian cancer cells. Collectively this study introduces a new and highly effective class of guanidinium-rich cell-penetrating transporters and methodology for their single-step conjugation to drugs and probes, and demonstrates that the resulting drug/probe-conjugates readily enter cells, outperforming previously reported guanidinium-rich oligocarbonates and peptide transporters.

N-Succinimidyl guanidinomethyl iodobenzoate protein radiohalogenation agents: influence of isomeric substitution on radiolabeling and target cell residualization.

INTRODUCTION: N-succinimidyl 4-guanidinomethyl-3-[(*)I]iodobenzoate ([(*)I]SGMIB) has shown promise for the radioiodination of monoclonal antibodies (mAbs) and other proteins that undergo extensive internalization after receptor binding, enhancing tumor targeting compared to direct electrophilic radioiodination. However, radiochemical yields for [(131)I]SGMIB synthesis are low, which we hypothesize is due to steric hindrance from the Boc-protected guanidinomethyl group ortho to the tin moiety. To overcome this, we developed the isomeric compound, N-succinimidyl 3-guanidinomethyl-5-[(131)I]iodobenzoate (iso-[(131)I]SGMIB) wherein this bulky group was moved from ortho to meta position. METHODS: Boc2-iso-SGMIB standard and its tin precursor, N-succinimidyl 3-((1,2-bis(tert-butoxycarbonyl)guanidino)methyl)-5-(trimethylstannyl)benzoate (Boc2-iso-SGMTB), were synthesized using two disparate routes, and iso-[*I]SGMIB synthesized from the tin precursor. Two HER2-targeted vectors - trastuzumab (Tras) and a nanobody 5F7 (Nb) - were labeled using iso-[(*)I]SGMIB and [(*)I]SGMIB. Paired-label internalization assays in vitro with both proteins, and biodistribution in vivo with trastuzumab, labeled using the two isomeric prosthetic agents were performed. RESULTS: When the reactions were performed under identical conditions, radioiodination yields for the synthesis of Boc2-iso-[(131)I]SGMIB were significantly higher than those for Boc2-[(131)I]SGMIB (70.7±2.0% vs 56.5±5.5%). With both Nb and trastuzumab, conjugation efficiency also was higher with iso-[(131)I]SGMIB than with [(131)I]SGMIB (Nb, 33.1±7.1% vs 28.9±13.0%; Tras, 45.1±4.5% vs 34.8±10.3%); however, the differences were not statistically significant. Internalization assays performed on BT474 cells with 5F7 Nb indicated similar residualizing capacity over 6h; however, at 24h, radioactivity retained intracellularly for iso-[(131)I]SGMIB-Nb was lower than for [(125)I]SGMIB-Nb (46.4±1.3% vs 56.5±2.5%); similar results were obtained using Tras. Likewise, a paired-label biodistribution of Tras labeled using iso-[(125)I]SGMIB and [(131)I]SGMIB indicated an up to 22% tumor uptake advantage at later time points for [(131)I]SGMIB-Tras. CONCLUSION: Given the higher labeling efficiency obtained with iso-SGMIB, this residualizing agent might be of value for use with shorter half-life radiohalogens.

Synthesis and structure-activity relationships of new carbonyl guanidine derivatives as novel dual 5-HT2B and 5-HT7 receptor antagonists. Part 2.

We previously reported that the novel dual 5-HT2B and 5-HT7 receptor antagonist N-(9-hydroxy-9H-fluorene-2-carbonyl)guanidine (4) exerted a suppressing effect on 5-HT-induced dural protein extravasation in guinea pigs. To develop a synthetic strategy, we performed docking studies of lead compound 4 bound to 5-HT2B and 5-HT7 receptors, and observed that the carbonyl guanidine group forms a tight interaction network with an active center Asp (D135:5-HT2B, D162:5-HT7), Tyr (Y370:5-HT2B, Y374:5-HT7) and aromatic residue (W131:5-HT2B, F158:5-HT7). Based on molecular modeling results, we optimized the substituents at the 5- to 8-position and 9-position of the fluorene ring and identified N-(diaminomethylene)-9-hydroxy-9-methyl-9H-fluorene-2-carboxamide (24a) exhibits potent affinity for 5-HT2B (Ki=4.3 nM) and 5-HT7 receptor (Ki=4.3 nM) with high selectivity over 5-HT2A, 5-HT2C, α1, D2 and M1 receptors. Compound 24a reversed the hypothermic effect of 5-carboxamidotryptamine (5-CT) in mice and also showed a suppressing effect on 5-HT-induced dural protein extravasation in guinea pigs when orally administered at 30 mg/kg. Compound 24a is therefore a promising candidate for a novel class of anti-migraine agent without any adverse effects.

Binding of organic dyes with human serum albumin: a single-molecule study.

Kinetics of binding of dyes at different sites of human serum albumin (HSA) has been studied by single-molecule spectroscopy. The protein was immobilized on a glass surface. To probe different binding sites (hydrophobic and hydrophilic) two dyes, coumarin 153 (C153, neutral) and rhodamine 6G (R6G, cationic) were chosen. For both the dyes, a major (ca. 96-98%) and minor (ca. 3%) binding site were detected. Rate constants of association and dissociation were simultaneously determined from directly measuring fluctuations in fluorescence intensity (τ(off) and τ(on)) and from this the equilibrium (binding) constants were calculated. Fluorescence lifetimes at individual sites were obtained from burst-integrated lifetime analysis. Distributions of lifetime histograms for both the probes (C153 and R6G) exhibit two maxima, which indicates the presence of two binding domains in the protein. Unfolding of the protein has been studied by adding guanidinium hydrochloride (GdnHCl) to the solution. It is observed that addition of GdnHCl affects the dissociation and association kinetics and hence, binding equilibrium of the association of C153. However, the effect of binding of R6G is not affected much. It is proposed that GdnHCl affects the hydrophobic binding sites more than the hydrophilic site.

Engineered modular recombinant transporters: application of new platform for targeted radiotherapeutic agents to alpha-particle emitting 211 At.

PURPOSE: To generate and evaluate a modular recombinant transporter (MRT) for targeting 211 At to cancer cells overexpressing the epidermal growth factor receptor (EGFR). METHODS AND MATERIALS: The MRT was produced with four functional modules: (1) human epidermal growth factor as the internalizable ligand, (2) the optimized nuclear localization sequence of simian vacuolating virus 40 (SV40) large T-antigen, (3) a translocation domain of diphtheria toxin as an endosomolytic module, and (4) the Escherichia coli hemoglobin-like protein (HMP) as a carrier module. MRT was labeled using N-succinimidyl 3-[211 At]astato-5-guanidinomethylbenzoate (SAGMB), its 125 I analogue SGMIB, or with 131 I using Iodogen. Binding, internalization, and clonogenic assays were performed with EGFR-expressing A431, D247 MG, and U87MG.wtEGFR human cancer cell lines. RESULTS: The affinity of SGMIB-MRT binding to A431 cells, determined by Scatchard analysis, was 22 nM, comparable to that measured before labeling. The binding of SGMIB-MRT and its internalization by A431 cancer cells was 96% and 99% EGFR specific, respectively. Paired label assays demonstrated that compared with Iodogen-labeled MRT, SGMIB-MRT and SAGMB-MRT exhibited more than threefold greater peak levels and durations of intracellular retention of activity. SAGMB-MRT was 10-20 times more cytotoxic than [211 At]astatide for all three cell lines. CONCLUSION: The results of this study have demonstrated the initial proof of principle for the MRT approach for designing targeted alpha-particle emitting radiotherapeutic agents. The high cytotoxicity of SAGMB-MRT for cancer cells overexpressing EGFR suggests that this 211 At-labeled conjugate has promise for the treatment of malignancies, such as glioma, which overexpress this receptor.

Pharmacokinetic properties of TDP4815 after single intravenous and oral administrations to rat, rabbit, monkey, dog and in vitro drug metabolism.

The pharmacokinetics of TDP4815 was evaluated in rats, rabbits, dogs and monkeys. After intravenous administration, TDP4815 achieved C(O) of 3255 ng/ml in rats at 5 mg/kg, 9066 ng/ml in rabbits and 7858 ng/ml in monkeys at 6 mg/kg, and 4457 ng/ml in dogs at 3 mg/kg. The clearance (C(L)) was 3105, 1692, 835 and 640 ml/h/kg in rats, rabbits, monkeys and dogs, respectively. The volume of distribution (V(Z)) was more than 3861 ml/kg in all species, except 1915 ml/kg in monkeys. The oral bioavailability was rabbit >rat> monkey compared at 100 mg/kg, but it was much higher in dogs (>64%) after oral administrations. The calculated intrinsic clearance data suggested that the clearance of dog and human was restricted by binding to the plasma protein, and the clearance of rat and monkey was dependent on both the free fraction of plasma protein binding and the liver blood flow rate. The unbound hepatic intrinsic clearance of monkey was close to its C(L) suggesting that the hepatic clearance was an important excretion in monkeys. The poor oral bioavailability in the monkey may be related to the extensive glucuronidation. The V(Z).kg and C(L).kg in test species showed good correlation with the animal body weights (R(2)=0.87 and 0.96).

Guanidinylated neomycin delivers large, bioactive cargo into cells through a heparan sulfate-dependent pathway.

Facilitating the uptake of molecules into living cells is of substantial interest for basic research and drug delivery applications. Arginine-rich peptides have been shown to facilitate uptake of high molecular mass cargos into cells, but the mechanism of uptake is complex and may involve multiple receptors. In this report, we show that a derivative of the aminoglycoside antibiotic neomycin, in which all of the ammonium groups have been converted into guanidinium groups, can carry large (>300 kDa) bioactive molecules across cell membranes. Delivery occurs at nanomolar transporter concentrations and under these conditions depends entirely on cell surface heparan sulfate proteoglycans. Conjugation of guanidinoneomycin to the plant toxin saporin, a ribosome-inactivating agent, results in proteoglycan-dependent cell toxicity. In contrast, an arginine-rich peptide shows both heparan sulfate-dependent and -independent cellular uptake. The high selectivity of guanidinoneomycin for heparan sulfate suggests the possibility of exploiting differences in proteoglycan compositions to target delivery to different cell types.

Highly efficient, nonpeptidic oligoguanidinium vectors that selectively internalize into mitochondria.

Oligoguanidinium-based cell delivery systems have gained broad interest in the drug delivery field since one decade ago. Thus, arginine-containing peptides as Tat or Antp, oligoarginine peptides, and derived peptoids have been described as shuttles for delivering nonpermeant drugs inside cancer cells. Herein we report a new family of tetraguanidinium cell penetrating vectors efficiently internalized in human tumor cells. Their high internalization, studied by confocal microscopy and flow cytometry, as well as their specific accumulation in mitochondria makes these new vectors likely vehicles for the targeted delivery of anticancer drugs to mitochondria.

Cellular uptake of aminoglycosides, guanidinoglycosides, and poly-arginine.

Aminoglycosides (including neomycin B and tobramycin) exhibit poor uptake by eukaryotic cell lines. When the amines of these natural products are converted into guanidine groups, their cellular uptake is dramatically enhanced. We have synthesized BODIPY-containing aminoglycosides and guanidinoglycosides to evaluate their cellular uptake properties. Fluorescence activated cell sorting (FACS) and fluorescence microscopy are used to compare the membrane translocation and the cellular localization of these compounds. Upon guanidinylation, the cellular uptake efficiencies of tobramycin and neomycin B are enhanced by 10-fold and 20-fold, respectively. Guanidino-neomycin B exhibits a highly efficient uptake, superior to a fluorescent poly-arginine peptide. Interestingly, the cellular uptake of this common transduction peptide is inhibited by guanidine-neomycin B, suggesting a similar uptake mechanism for both the arginine-rich peptides and the guanidinoglycosides.

Novel binding and efficient cellular uptake of guanidine-based peptide nucleic acids (GPNA).

Incorporation of a guanidine functional group into the PNA backbone facilitates cellular uptake of PNA into mammalian cells with efficiency comparable to that of the TAT transduction domain. The modified PNA recognizes and binds to the complementary DNA strand in accordance with Watson-Crick recognition rules. However, unlike polypyrimidine PNA which binds to DNA in 2:1 stoichiometry, the modified PNA binds to complementary DNA in a 1:1 ratio to form a highly stable duplex.

N-succinimidyl 3-[211At]astato-4-guanidinomethylbenzoate: an acylation agent for labeling internalizing antibodies with alpha-particle emitting 211At.

The objective of this study was to develop a method for labeling internalizing monoclonal antibodies (mAbs) such as those reactive to the anti-epidermal growth factor receptor variant III (EGFRvIII) with the alpha-particle emitting radionuclide (211)At. Based on previous work utilizing the guanidine-containing acylation agent, N-succinimidyl 4-guanidinomethyl-3-[(131)I]iodobenzoate ([(131)I]SGMIB), we have now investigated the potential utility of its astato analogue for labeling the anti-EGFRvIII mAb L8A4. N-succinimidyl 3-[(211)At]astato-4-guanidinomethylbenzoate ([(211)At]SAGMB) in its Boc-protected form was prepared from a tin precursor in 61.7 +/- 13.1% radiochemical yield, in situ deprotected to [(211)At]SAGMB, which was coupled to L8A4 in 36.1 +/- 1.9% yield. Paired-label internalization assays demonstrated that tumor cell retention of radioactivity for L8A4 labeled using [(211)At]SAGMB was almost identical to L8A4 labeled using [(131)I]SGMIB, and 3-4-fold higher than for mAb radioiodinated using Iodogen. Paired-label biodistribution of L8A4 labeled using [(211)At]SAGMB and [(131)I]SGMIB in athymic mice hosting U87MGdeltaEGFR xenografts resulted in identical uptake of both (211)At and (131)I in tumor tissues over 24 h. Although higher levels of (211)At compared with (131)I were sometimes seen in tissues known to sequester free astatide, these (211)At/(131)I uptake ratios were considerably lower than those seen with other labeling methods. These results suggest that [(211)At]SAGMB may be a useful acylation agent for labeling internalizing mAbs with (211)At.

Guanidine transport across the apical and basolateral membranes of human intestinal Caco-2 cells is mediated by two different mechanisms.

The functional characteristics of the intestinal absorption and secretion of guanidine as a model of a nutritionally and metabolically essential organic cation were examined in the Caco-2 human intestinal cell line. Both apical and basolateral transport of [14C]-guanidine were studied using Caco-2 cells grown on polycarbonate permeable membranes. The basolateral-to-apical flux of [14C]-guanidine (i.e., its secretion) was quantitatively higher than the apical-to-basolateral transport (i.e., its absorption). When Na+ was replaced by K+ or Li+, both apical and basolateral accumulation were significantly inhibited. Studies using the cell monolayers and apical membrane vesicles obtained from Caco-2 cells showed a potential-independent mechanism of guanidine apical uptake and efflux. Conversely, basolateral uptake and efflux were membrane potential dependent. Kinetic analysis revealed that both saturable and nonsaturable mechanisms accounted for the apical and basolateral accumulations. The [14C]-guanidine efflux from cells through the apical and basolateral membranes was significantly reduced at 4 degrees C, suggesting carrier-mediated mechanisms. Moreover, the apical efflux was stimulated by an inwardly directed H+ gradient. Influx and efflux of [14C]-guanidine were unaffected by the presence of tetraethylammonium, cimetidine or decynium-22 in the donor compartment. Only quinine significantly reduced [14C]-guanidine entrance through apical and basolateral membranes and its exit through the basolateral membrane. In conclusion, our results suggest that the influx and the efflux through the apical membrane is mediated by different transporters, whereas transport across the basolateral membrane is mediated by a member of the organic cation transporter family with high affinity for guanidine.

Novel potent 5-HT(3) receptor ligands based on the pyrrolidone structure: synthesis, biological evaluation, and computational rationalization of the ligand-receptor interaction modalities.

Novel conformationally constrained derivatives of classical 5-HT(3) receptor antagonists were designed and synthesized with the aim of probing the central 5-HT(3) receptor recognition site in a systematic way. The newly-synthesized compounds were tested for their potential ability to inhibit [(3)H]granisetron specific binding to 5-HT(3) receptor in rat cortical membranes. These studies revealed subnanomolar affinity in some of the compounds under study. The most potent ligand in this series was found to be quinuclidine derivative (S)-7i, which showed an affinity comparable with that of the reference ligand granisetron. The potential 5-HT(3) agonist/antagonist activity of some selected compounds was assessed in vitro on the 5-HT(3) receptor-dependent [(14)C]guanidinium uptake in NG 108-15 cells. Both of the tropane derivatives tested in this functional assay (7a and 9a) showed antagonist properties, while the quinuclidine derivatives studied [the enantiomers of compounds 7i, 8g, and 9g, and compound (R)-8h] showed a full range of intrinsic efficacies. Therefore, the functional behavior of these 5-HT(3) receptor ligands appears to be affected by the structural features of both the azabicyclo moiety and the heteroaromatic portion. In agreement with the data obtained on NG 108-15 cells, investigations on the 5-HT(3) receptor-dependent Bezold-Jarisch reflex in urethane-anaesthetized rats confirmed the 5-HT(3) receptor antagonist properties of compounds 7a and (S)-7i showing for these compounds ID(50) values of 2.8 and 181 microg/kg, respectively. Finally, compounds 7a, (S)-7i and 9a (at the doses of 0.01, 1.0, and 0.01 mg/kg ip, respectively) prevented scopolamine-induced amnesia in the mouse passive avoidance test suggestive of a potential usefulness in cognitive disorders for these compounds. Qualitative and quantitative structure-affinity relationship studies were carried out by means of theoretical descriptors derived on a single structure and ad-hoc defined size and shape descriptors (indirect approach). The results showed to be useful in capturing information relevant to ligand-receptor interaction. Additional information derived by the analysis of the energy minimized 3-D structures of the ligand-receptor complexes (direct approach) suggested interesting mechanistic and methodological considerations on the binding mode multiplicity at the 5-HT(3) receptors and on the degree of tolerance allowed in the alignment of molecules for the indirect approach, respectively.

A polar substituent-containing acylation agent for the radioiodination of internalizing monoclonal antibodies: N-succinimidyl 4-guanidinomethyl-3-[131I]iodobenzoate ([131I]SGMIB).

The objective of this study was to develop an acylation agent for the radioiodination of monoclonal antibodies that would maximize retention of the label in tumor cells following receptor- or antigen-mediated internalization. The strategy taken was to add a polar substituent to the labeled aromatic ring to impede transport of labeled catabolites across lysosomal and cell membranes after antibody degradation. Preparation of unlabeled N-succinimidyl 4-guanidinomethyl-3-iodobenzoate (SGMIB) was achieved in six steps from 3-iodo-4-methylbenzoic acid. Preparation of 4-guanidinomethyl-3-[131I]iodobenzoic acid from the silicon precursor, 4-(N1,N2-bis-tert-butyloxycarbonyl)guanidinomethyl-3-trimethylsilylbenzoic acid proceeded in less than 5% radiochemical yield. A more successful approach was to prepare [131I]SGMIB directly from the tin precursor, N-succinimidyl 4-(N1,N2-bis-tert-butyloxycarbonyl)guanidinomethyl-3-trimethylstannylbenzoate, which was achieved in 60-65% radiochemical yield. A rapidly internalizing anti-epidermal growth factor receptor variant III antibody L8A4 was labeled using [131I]SGMIB in 65% conjugation efficiency and with preservation of immunoreactivity. Paired-label in vitro internalization assays demonstrated that the amount of radioactivity retained in cells after internalization for L8A4 labeled with [131I]SGMIB was 3-4-fold higher than that for L8A4 labeled with 125I using either Iodogen or [125I]SIPC. Catabolite assays documented that the increased retention of radioiodine in tumor cells for antibody labeled using [131I]SGMIB was due to positively charged, low molecular weight species. These results suggest that [131I]SGMIB warrants further evaluation as a reagent for labeling internalizing antibodies.

Functional characterization of organic cation drug transport in the pigmented rabbit conjunctiva.

PURPOSE: To characterize carrier-mediated organic cation drug transport in the rabbit conjunctiva. METHODS: The transport of [14C]guanidine, the model substrate, in the excised pigmented rabbit conjunctiva was evaluated in the modified Ussing chamber. Tetraethylammonium (TEA) transport also was investigated to determine substrate specificity. RESULTS: The apparent permeability coefficient for guanidine and TEA in the mucosal-to-serosal (ms) direction was 5.4 and 49.6 times greater than that in the serosal-to-mucosal (sm) direction, respectively. Guanidine transport in the ms (but not sm) direction revealed temperature and concentration dependency over 0.02 to 10 mM with an apparent Michaelis-Menten constant of 3.1 mM and a maximal flux of 11.4 nmol/(cm2 x h). Net guanidine transport measured at 0.1 mM across the conjunctiva was decreased by 71% or 82%, respectively, on the addition of 1 microM valinomycin (a K+ ionophore) in both bathing fluids or in a high K+ buffer in the mucosal fluid. Interestingly, net guanidine transport was reduced, rather than enhanced, by 63% upon acidifying the mucosal bathing fluid. By contrast, net guanidine transport was not affected by the serosal presence of 0.5 mM ouabain (a Na+, K+-ATPase inhibitor), by the mucosal and serosal presence of 0.1 microM monensin (a Na+ ionophore) or 0.3 microM carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone (FCCP, a H+ ionophore). Guanidine transport in the ms direction was polyspecific, as indicated by the 48% to 82% inhibition by structurally diverse amines. In particular, guanidine ms transport was inhibited by the antiglaucoma drugs dipivefrine (72%), brimonidine (70%), and carbachol (78%). CONCLUSIONS: A carrier-mediated organic cation transport process appears to exist in the conjunctiva, mediating the absorption of organic amines, including certain amine-type ophthalmic drugs. This process may be driven by an inside-negative apical membrane potential difference.

Pharmacologic and molecular discrimination of I2-imidazoline receptor subtypes.

I2-imidazoline receptors (I2-IR) are characterized by their high affinity for imidazolines and guanidines and medium affinity for imidazolidines. The differential recognition of I2-IR by amiloride led to subtype these sites as amiloride-sensitive (I2A-IR) and amiloride-insensitive (I2B-IR). I2-IR labeled with [3H]idazoxan or [3H]2-BFI in the rabbit cerebral cortex (I2A-IR) displayed higher affinities for amiloride and amiloride analogs than in the rat cerebral cortex (I2B-IR). Other drugs tested displayed biphasic curves in competition experiments, indicating the existence of high and low affinity sites for both I2-IR subtypes. The drugs (+)- and (-)-medetomidine, bromoxidine, moxonidine, and clorgyline were more potent on the high and/or low affinity sites of I2B-IR than on I2A-IR. Preincubation (30 min at 25 degrees C) with 10(-6) M isothiocyanatobenzyl imidazoline (IBI) or with 10(-6) M clorgyline reduced by 40% and 26%, respectively, the binding of [3H]2-BFI to I2B-IR, but it did not alter the binding of the radioligand to I2A-IR. These results indicated that the I2-IR subtypes differ in their pharmacologic profiles and in the nature of the imidazoline binding site involved in clorgyline and IBI alkylation. In rat cortical membranes, western blot detection of immunoreactive imidazoline receptor proteins revealed a double band of approximately 29/30 kD and three less intense bands of approximately 45, approximately 66, and approximately 85 kD. In rabbit cortical membranes the antibody detected proteins of approximately 30, approximately 57, approximately 66, and approximately 85 kD. It is suggested that I2-IR may be related to more than one receptor protein and that I2-IR subtypes differ in the nature of the proteins implicated.