Synthesis and biological activity of oxo-7H-benzo[e]perimidine-4-carboxylic acid derivatives as potent, nonpeptide corticotropin releasing factor (CRF) receptor antagonists.

A novel series of derivatives of oxo-7H-benzo[e]perimidine-4-carboxylic acid (I) potently displaced radioligand binding of 125I-CRF to both CRF1 and CRF2 receptors. The members of this series antagonized CRF-stimulated cAMP formation and CRF-stimulated corticotropin release from rat pituitary in vivo. These are the first nonpeptide antagonists to show activity at both CRF1 and CRF2 receptors.

Double tagging recombinant A1- and A2A-adenosine receptors with hexahistidine and the FLAG epitope. Development of an efficient generic protein purification procedure.

An expression plasmid for mammalian cells (CLDN10B) has been modified to add nucleotides encoding hexahistidine and the FLAG peptide (H/F) to cDNAs. The new mammalian expression plasmid has been named pDoubleTrouble (pDT). The plasmid and a recombinant baculovirus were used to produce native-and H/F-human A1 and A2A adenosine receptors, optimally expressed in CHO-K1 and Sf9 cells, respectively. Binding to recombinant H/F-A1 receptors (Bmax = 30 pmol/mg protein) was characterized using [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]CPX) and 125I-N6-aminobenzyladenosine (125I-ABA). Binding to H/F-A2A receptors (Bmax = 48 pmol/mg protein) was characterized using [3H]5'-N-ethylcarboxamidoadenosine ([3H]NECA) and [3H]2-[4-(2-carboxyethyl)phenethylamino]-NECA ([3H]CGS21680). By comparison to native receptors, the addition of H/F to the amino termini of these receptors had no effect on the binding affinities cyclic AMP accumulation in intact cells was not affected by the H/F extension. Anti-FLAG and Ni-nitrilotriacetic acid affinity chromatography resulted in high yield ( >50% overall recovery) of nearly homogeneous deglycosylation with N-glycosidase F. We anticipate that pDT will be generally useful for facilitating the purification in high yield of recombinant receptors and other proteins by single or sequential affinity chromatography steps.

Photoaffinity labeling with 2(-)[2-(4-azido-3(-)[125I]- iodophenyl)ethylamino]adenosine and autoradiography with 2(-)[2-(4-amino-3(-)[125I]iodophenyl)ethylamino]adenosine of A2a adenosine receptors in rat brain.

The A2a adenosine receptor agonist 2(-)[2-(4-amino-3- iodophenyl)ethylamino]adenosine is a potent coronary vasodilator. The corresponding radioiodinated ligand, [125I]APE, discriminates between high- and low-affinity conformations of A2a adenosine receptors. In this study, [125I]APE was used for rapid (24-h) autoradiography in rat brain sections. The pattern of [125I]APE binding is consistent with that expected of an A2a-selective radioligand. It is highest in striatum, nucleus accumbens, and olfactory tubercle, with little binding to cortex and septal nuclei. Specific [125I]APE binding to these brain regions is abolished by 1 microM 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680) but is little affected by 100 nM 8-cyclopentyl-1,3-dipropylxanthine. Conversion of [125I]APE to the corresponding arylazide results in [125I]AzPE. The rank-order potency of compounds to compete for [125I]AzPE binding in the dark is CGS-21680 > D-(R)-N6-phenylisopropyladenosine > N6- cyclopentyladenosine, indicating that it also is an A2a-selective ligand. Specific photoaffinity labeling by [125I]AzPE of a single polypeptide (42 kDa) corresponding to A2a adenosine receptors is reduced 55 +/- 4% by 100 microM guanosine 5'-O-(3-thiotriphosphate) and 91 +/- 1.3% by 100 nM CGS-21680. [125I]APE and [125I]AzPE are valuable new tools for characterizing A2a adenosine receptors and their coupling to GTP-binding proteins by autoradiography and photoaffinity labeling.

Evidence for regulated coupling of A1 adenosine receptors by phosphorylation in Zucker rats.

Studies were designed to find the molecular basis for previous observations that lipolysis is less active and A1 adenosine receptor signaling is more active in adipocytes from obese than from lean Zucker rats. With quantitative immunoblot procedures for detection, Gi alpha 1 and Gs alpha 45 levels were found anomalously low in obese compared with lean membranes (50 and 30%, respectively), but other G alpha subunit levels were normal. However, the sensitivity of the receptor-Gi protein to GTP was about 5- to 10-fold higher in obese compared with lean membranes when assessed from 1) the ability of GTP to inhibit forskolin-stimulated adenylyl cyclase in the presence of an adenosine receptor agonist and 2) the ability of a nonhydrolyzable guanine nucleotide analogue to alter A1 adenosine receptor agonist binding. Alkaline phosphatase treatment of isolated adipocyte membranes from obese but not lean animals decreased guanine nucleotide sensitivity of agonist binding. Surprisingly, solubilized adipocyte A1 adenosine receptors from all animals exhibited the same high sensitivity to guanine nucleotides as that of intact obese membranes, and this high sensitivity could be decreased 20-fold by treatment with alkaline phosphatase. These data suggest that protein phosphorylation may regulate coupling of the A1 adenosine receptor in rat adipocyte membranes.

Comparison of A4 and A2a binding sites in striatum and COS cells transfected with adenosine A2a receptors.

A putative A4 adenosine receptor is characterized by a distinct structure activity profile of compounds in competition for [3H]2-phenylaminoadenosine ([3H]CV 1808) binding sites on rat brain membranes assayed at 4 degrees C. We now confirm that A4 binding sites can be demonstrated on ice-cold membranes of rat striatum and demonstrate a similar binding site on COS cells transfected with rat A2a adenosine receptors (COS/A2a). The characteristic A4 potency order is: CV 1808 > [1R-(1 alpha, 2 alpha, 3 beta, 5 beta)]-3-(2,6-diamino-N2-(3-carbethoxyphenyl)-9 H-purin-9-yl)-5'-(N-ethylcarbamoyl)-1,2-cyclopentanediol (CGS 22988) >> 5'-N-ethylcarboxamidoadenosine (NECA) > or = 2-[4-(2- carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680); 9-chloro-2-(2-furyl)[1,2,4]-triaolo[1,5-c]-quinazolin-5-a min e (CGS 15943) only partially inhibits binding at 1 microM. If [3H]CGS 21680 is used for ice-cold assays, or if either [3H]CV 1808 or [3H]CGS 21680 are used for assays at 21 degrees C, the potency order of competing compounds changes markedly and becomes characteristic of A2a adenosine receptor binding sites; CGS 15943 > or = CGS 21680 congruent to NECA > CGS 22988 > or = CV 1808. Binding of [3H]CGS 21680, but not [3H]CV 1808, is significantly enhanced by the pore-forming antibiotic, alamethicin. Guanosine 5'-O-(3-thiotriphosphate) decreases the binding of both radioligands to striatal membranes at 21 degrees C significantly more than to membranes on ice.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of two affinity states of adenosine A2a receptors with a new radioligand, 2-[2-(4-amino-3-[125I]iodophenyl)ethylamino]adenosine.

Adenosine analogs substituted in the 2-position with arylamino groups have been found to have high affinity and selectivity for A2a adenosine receptors. Two such compounds, 2-[2-(4-aminophenyl)ethylamino]adenosine and 2-[2-4-amino-3-iodophenyl)ethylamino]adenosine (I-APE), were synthesized and found to be potent coronary vasodilators (ED50 < 3 nm). These compounds bind weakly to A1 adenosine receptors of rat cortex (Ki > 150 nM). 125I-APE was synthesized and the new radioligand was found to bind to two affinity states of rat striatal A2a adenosine receptors (Kd = 1.3 +/- 0.1 nM and 19 +/- 4.5 nM). The high affinity site represents a previously unrecognized small (15-20%) fraction of A2a adenosine receptors coupled to G proteins. Guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) reduces specific binding of 125I-APE half-maximally at a concentration of 45 +/- 2 nM. [3H]CGS21680 also binds to two affinity states of A2a receptors on striatal membranes (Kd = 3.9 +/- 0.9 and 51 +/- 5.5 nM), although in previous studies single Kd values ranging from 5 to 15 nM have been reported. This high affinity site is substantiated by the finding that the IC50 of CGS21680 in competition with 125I-APE binding to striatal membranes is shifted leftward in membranes diluted for 4 min before filtration, to selectively dissociate radioligand from low affinity receptors. Assuming that agonist radioligands bind to both coupled and uncoupled forms of striatal A2a adenosine receptors, we could simulate with the computer the finding that the decrease in specific binding induced by GTP gamma S (100 microM) is variable and depends on radioligand concentration, ranging from 20 to 90%. Unlike 125I-APE, [3H]CGS21680 is charged at physiological pH, and treatment of membranes with the pore-forming antibiotic alamethicin uncovers cryptic [3H]CGS21680 but not 125I-APE binding sites. We conclude that the GTP gamma S-sensitive high affinity form of the A2a adenosine receptor can be preferentially labeled by 125I-APE, due to both its high specific activity and its physicochemical properties. Possible functional manifestations of poor coupling of A2a adenosine receptors to G proteins are discussed.

Purification and partial amino acid sequence of a mu opioid receptor from rat brain.

A rat brain opioid receptor protein was isolated by binding [epsilon-biotinyl-Lys32] beta-endorphin to membranes, solubilizing the receptor-ligand (R.L) complex with deoxycholate-lysophosphatidylcholine and purifying on immobilized streptavidin and wheat germ agglutinin. The purified glycoprotein had a molecular mass of 60-70 kDa. Recovery of this protein was blocked by the nonselective opioid antagonist naloxone and the highly mu-selective agonist [D-Ala2,N-methyl-Phe4,Glyol5]-enkephalin but not by the highly delta-selective agonist [D-Pen2,4'-Cl-Phe4,D-Pen5]enkephalin when these compounds were added as competitors at the binding step. The 60-70-kDa receptor protein co-purified through the streptavidin column with 40-kDa protein recognized by anti-Gi alpha antibodies. GTP and Na+ influenced dissociation of the solubilized R.125I-L complex and elution of the receptor and G protein from streptavidin in fashions consistent with the pharmacology of mu-opioid receptors. A 23-amino acid residue sequence from the purified receptor differs at 4 positions from a similar sequence in the murine delta-opioid receptor and is encoded within a novel rat brain cDNA isolated by polymerase chain reaction with oligonucleotide primers related to the murine delta-opioid receptor gene.

Genomic analysis and expression patterns reveal distinct genes for endothelial and brain nitric oxide synthase.

Constitutively active nitric oxide synthases (NOS) are a unique class of NADPH-dependent, calcium/calmodulin-dependent enzymes that catalyze the conversion of L-arginine to nitric oxide and L-citrulline. However, little is known about the molecular similarities or differences between the two prototypical constitutive NOS enzymes, endothelial NOS (ECNOS) and brain NOS (bNOS). The aims of this study were to begin characterizing the gene structure and tissue distribution of messenger RNAs (mRNAs) for ECNOS and bNOS and to examine the immunological resemblance of the proteins by Western blotting. Full-length complementary DNAs (cDNAs) encoding bovine ECNOS and rat bNOS hybridized, under high stringency, to different-sized fragments of endonuclease-digested bovine, rat, and human genomic DNA. In addition, more than one fragment was detected with both cDNAs, suggesting that ECNOS and bNOS genes contained multiple introns. Tissue distribution of ECNOS mRNA (4.4 kb) and bNOS mRNA (9.5 kb) in the rat was detected by Northern blotting. Patterns among tissue extracts were strikingly different, with ECNOS mRNA being most abundant in aorta, heart, lung, kidney, adrenal gland, spinal cord, and urogenital tissues and bNOS mRNA most prominent in brain regions, intestine, stomach, spinal cord, adrenal gland, and aorta. Interestingly, ECNOS cDNA detected two equally abundant RNA transcripts (4.4 and 4.0 kb) in most brain regions tested, suggesting an alternative splicing of the ECNOS pre-mRNA. Western blotting, using an ECNOS monoclonal antibody, recognized ECNOS protein from native bovine endothelial cells, cultured bovine endothelial cells, and COS cells transfected with ECNOS cDNA but did not recognize purified bNOS.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and quantification of Gi-type GTP-binding proteins that copurify with a pituitary somatostatin receptor.

Somatostatin (SRIF) receptors of GH4C1 cells occupied with biotinyl-NH-[Leu8,D-Trp22,Tyr25] somatostatin28 (bio-S28) have been affinity purified over streptavidin affinity columns (Eppler, C. M., Zysk, J. R., Corbett, M., and Shieh, H.-M. (1992) J. Biol. Chem. 267, 15603-15612). This procedure results in the copurification of a single subtype of SRIF receptor (SSTR2) and associated guanine nucleotide-binding proteins (G proteins) that are coupled to these receptors. For accurate quantification it was necessary to: (i) use homogenous recombinant standards; (ii) accurately assess the purity of standards; (iii) determine recovery of G proteins during sample preparation and Western blotting; and (iv) account for cross-reactivity among antisera. Four pertussis toxin-sensitive G proteins were quantified with previously characterized polyclonal antisera. Gi alpha 1 also was measured with a novel, more sensitive monoclonal antibody (7H7). Go alpha and Gi alpha 2 but not Gi alpha 1 and Gi alpha 3 were detected in membrane extracts prepared from GH4C1 cells. In contrast, the G proteins copurified with SSTR2 receptors were predominantly Gi alpha 2 (50% of total G protein) and Gi alpha 3 (36% of total G protein), whereas Go alpha and Gi alpha 1 were negligible. G beta subunits also were detected. Silver staining confirmed the absence of a 39-kDa protein, corresponding to the M(r) of Go alpha associated with purified SRIF receptor-G protein complexes. These data suggest that SRIF receptors selectively couple to two G proteins, one of which is sparsely expressed in GH4C1 cells; the data conform to the notion that SRIF receptors discriminate between similar pertussis toxin-sensitive G proteins.

Evidence for enhanced inhibitory modulation by cyclooxygenase products of noradrenergic neurotransmission in the mesenteric vasculature of young spontaneously hypertensive rats.

The effects of cyclooxygenase inhibition by indomethacin and meclofenamate on pre- and postjunctional aspects of noradrenergic neurotransmission were determined in mesenteric vascular preparations from 4- to 6-week-old spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto rats (WKYs). Perfusion pressure responses to periarterial nerve stimulation and to exogenous norepinephrine (NE) were significantly greater in SHR than in WKY preparations, whereas fractional NE overflow was equivalent between the two strains. Indomethacin and meclofenamate enhanced perfusion pressure responses to periarterial nerve stimulation in both strains. Fractional NE overflow was significantly enhanced by indomethacin but only at 14 Hz in SHR preparations, whereas it was unaffected in WKY preparations. The combination of indomethacin and cocaine resulted in a significant enhancement of perfusion pressure responses to periarterial nerve stimulation in both strains that was significantly greater than that produced by either drug alone. This effect was significantly greater in SHR than in WKY preparations. This combination also resulted in a significant enhancement of responses to exogenous NE in both strains. Fractional NE overflow was significantly increased in SHR preparations in the presence of the combination of cocaine and indomethacin, whereas it remained unaltered in WKY preparations. These findings suggest that a cyclooxygenase product exerts both pre- and postjunctional inhibitory effects on vascular noradrenergic neurotransmission that differ in these two strains of rats. The prejunctional inhibitory effect of this cyclooxygenase product was observed only in SHR preparations and was especially evident in the presence of cocaine.