A small-molecule inhibitor of integrin alpha2 beta1 introduces a new strategy for antithrombotic therapy.

Interaction of blood platelets with vascular collagen is an initiating event in haemostasis and thrombus formation. Based on molecular modelling of human integrin alpha2I domain and cell-based screening assays we have developed sulfonamide derivatives, a mechanistically novel class of molecules. These molecules show antiplatelet efficacy by selectively inhibiting alpha2beta1 integrin-mediated collagen binding. One sulfonamide derivative, named BTT-3016, showed inhibitory capacity in several assessments of human platelet interaction with collagen. It inhibited about 90% of the aggregation of gel-filtered magnesium-supplemented platelets and 70% of aggregation in PPACK-anticoagulated platelet-rich plasma when stimulated with collagen but not with ADP. The antiplatelet activity of BTT-3016 was dependent on alpha2beta1 integrin, since in collagen binding test BTT-3016 had no effect on the platelets derived from alpha2 integrin null mice. When tested in an in vivo model in mice, BTT-3016 clearly reduced thrombus formation on the vessel wall after vascular injury. Furthermore, BTT-3016 prolonged tail-bleeding time in a manner comparable to aspirin. We show that new alpha2beta1 inhibitors exert collagen-specific antiplatelet activity and regulate thrombus growth in vivo without compromising primary haemostasis more than aspirin. We suggest that the alpha2beta1 inhibiting strategy could be further developed for the prevention and treatment of arterial thrombosis.

Phenoxybenzamine binding reveals the helical orientation of the third transmembrane domain of adrenergic receptors.

Phenoxybenzamine (PB), a classical alpha-adrenergic antagonist, binds irreversibly to the alpha-adrenergic receptors (ARs). Amino acid sequence alignments and the predicted helical arrangement of the seven transmembrane (TM) domains suggested an accessible cysteine residue in transmembrane 3 of the alpha(2)-ARs, in position C(3.36) (in subtypes A, B, and C corresponding to amino acid residue numbers 117/96/135, respectively), as a possible site for the PB interaction. Irreversible binding of PB to recombinant human alpha(2)-ARs (90 nm, 30 min) reduced the ligand binding capacity of alpha(2A)-, alpha(2B)-, and alpha(2C)-AR by 81, 96, and 77%. When the TM3 cysteine, Cys(117), of alpha(2A)-AR was mutated to valine (alpha(2A)-C117V), the receptor became resistant to PB (inactivation, 10%). The beta(2)-AR contains a valine in this position (V(3.36); position number 117) and a cysteine in the preceding position (Cys(116)) and was not inactivated by PB (10 microm, 30 min) (inactivation 26%). The helical orientation of TM3 was tested by exchanging the amino acids at positions 116 and 117 of the alpha(2A)-AR and beta(2)-AR. The alpha(2A)-F116C/C117V mutant was resistant to PB (inactivation, 7%), whereas beta(2)-V117C was irreversibly inactivated (inactivation, 93%), confirming that position 3.36 is exposed to receptor ligands, and position 3.35 is not exposed in the binding pocket.

Molecular mechanism for agonist-promoted alpha(2A)-adrenoceptor activation by norepinephrine and epinephrine.

We present a mechanism for agonist-promoted alpha(2A)-adrenergic receptor (alpha(2A)-AR) activation based on structural, pharmacological, and theoretical evidence of the interactions between phenethylamine ligands and alpha(2A)-AR. In this study, we have: 1) isolated enantiomerically pure phenethylamines that differ both in their chirality about the beta-carbon, and in the presence/absence of one or more hydroxyl groups: the beta-OH and the catecholic meta- and para-OH groups; 2) used [(3)H]UK-14,304 [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine; agonist] and [(3)H]RX821002 [2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline; antagonist] competition binding assays to determine binding affinities of these ligands to the high- and low-affinity forms of alpha(2A)-AR; 3) tested the ability of the ligands to promote receptor activation by measuring agonist-induced stimulation of [(35)S]GTPgammaS binding in isolated cell membranes; and 4) used automated docking methods and our alpha(2A)-AR model to predict the binding modes of the ligands inside the alpha(2A)-AR binding site. The ligand molecules are sequentially missing different functional groups, and we have correlated the structural features of the ligands and ligand-receptor interactions with experimental ligand binding and receptor activation data. Based on the analysis, we show that structural rearrangements in transmembrane helix (TM) 5 could take place upon binding and subsequent activation of alpha(2A)-AR by phenethylamine agonists. We suggest that the following residues are important in phenethylamine interactions with alpha(2A)-AR: Asp113 (D(3.32)), Val114 (V(3.33)), and Thr118 (T(3.37)) in TM3; Ser200 (S(5.42)), Cys201 (C(5.43)), and Ser204 (S(5.46)) in TM5; Phe391 (F(6.52)) and Tyr394 (Y(6.55)) in TM6; and Phe411 (F(7.38)) and Phe412 (F(7.39)) in TM7.

Ligand recognition of serine-cysteine amino acid exchanges in transmembrane domain 5 of alpha2-adrenergic receptors by UK 14,304.

Ligand binding of UK 14,304 reveals notable species (i.e., human-rodent) and receptor-subtype differences of alpha2-adrenergic receptors (alpha2-ARs). To study the molecular basis of the selectivity of UK 14,304, we compared a series of conservative serine-cysteine exchange mutants at ligand-accessible positions in transmembrane domain 5 of the human and mouse alpha2A-ARs. UK 14,304 bound with approximately 200-fold higher affinity to the human alpha2A-AR wild-type receptor compared with the human alpha2A-ARSer201 mutant, but only an approximately fivefold difference was seen with the corresponding mouse alpha2A-AR variant. These effects of cysteine-serine exchanges only involved the agonist low-affinity forms of the receptors, as the affinity of [3H]UK 14,304 for the agonist high-affinity receptor populations was not influenced. The apparent affinities of a set of eight structurally diverse alpha2-AR ligands (six agonists and two antagonists) were not influenced significantly by the cysteine-serine exchanges (except for oxymetazoline and yohimbine, with up to nine- and eightfold differences in affinity, respectively). We conclude that position 201 (a) plays a primary role in determining observed subtype/species selectivity of UK 14,304 in competitive antagonist radioligand binding assays and (b) does not determine the subtype selectivity of chlorpromazine.

Three-dimensional models of alpha(2A)-adrenergic receptor complexes provide a structural explanation for ligand binding.

We have compared bacteriorhodopsin-based (alpha(2A)-AR(BR)) and rhodopsin-based (alpha(2A)-AR(R)) models of the human alpha(2A)-adrenengic receptor (alpha(2A)-AR) using both docking simulations and experimental receptor alkylation studies with chloroethylclonidine and 2-aminoethyl methanethiosulfonate hydrobromide. The results indicate that the alpha(2A)-AR(R) model provides a better explanation for ligand binding than does our alpha(2A)-AR(BR) model. Thus, we have made an extensive analysis of ligand binding to alpha(2A)-AR(R) and engineered mutant receptors using clonidine, para-aminoclonidine, oxymetazoline, 5-bromo-N-(4, 5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14,304), and norepinephrine as ligands. The representative docked ligand conformation was chosen using extensive docking simulations coupled with the identification of favorable interaction sites for chemical groups in the receptor. These ligand-protein complex studies provide a rational explanation at the atomic level for the experimentally observed binding affinities of each of these ligands to the alpha(2A)-adrenergic receptor.

Chloroethylclonidine and 2-aminoethyl methanethiosulfonate recognize two different conformations of the human alpha(2A)-adrenergic receptor.

The substituted cysteine-accessibility method and two sulfhydryl-specific reagents, the methane-thiosulfonate derivative 2-aminoethyl methanethiosulfonate (MTSEA) and the alpha(2)-adrenergic receptor (alpha(2)-AR) agonist chloroethylclonidine (CEC), were used to determine the relative accessibility of engineered cysteines in the fifth transmembrane domain of the human alpha(2A)-AR (Halpha2A). The second-order rate constants for the reaction of the receptor with MTSEA and CEC were determined with the wild type Halpha2A (cysteine at position 201) and receptor mutants containing accessible cysteines at other positions within the binding-site crevice (positions 197, 200, and 204). The rate of reaction of CEC was similar to that of MTSEA at residues Cys-197, Cys-201, and Cys-204. The rate of reaction of CEC with Cys-200, however, was more than 5 times that of MTSEA, suggesting that these compounds may interact with two different receptor conformations. MTSEA, having no recognition specificity for the receptor, likely reacts with the predominant inactive receptor conformation (R), whereas the agonist CEC may stabilize and react preferentially with the active receptor conformation (R*). This hypothesis was consistent with three-dimensional receptor-ligand models, which further suggest that alpha(2A)-AR activation may involve the clockwise rotation of transmembrane domain 5.

A series of 6-(omega-methanesulfonylthioalkoxy)-2-N-methyl- 1,2,3, 4-tetrahydroisoquinolines: cysteine-reactive molecular yardsticks for probing alpha2-adrenergic receptors.

A series of 6-(omega-methanesulfonylthioalkoxy)-2-N-methyl-1,2,3, 4-tetrahydroisoquinolines (7a-d) was prepared and characterized as SH-reactive molecular yardsticks useful in probing alpha2-adrenergic receptors. Rapid displacement of the methanesulfonyl group by a cysteine residue in dilute aqueous solution with concomitant formation of a disulfide conjugate was verified by MALDI-TOF mass spectrometric analysis of the reaction of 7a with a cysteine-containing decapeptide. 7a-d all showed a marked affinity for the three different variants of human alpha2-adrenergic receptors: H alpha(2A)wt, H alpha(2B)wt, and mutant H alpha(2A)Ser201Cys197. However, only the mutated receptor (H alpha(2A)Ser201Cys197) was irreversibly inactivated, and the extent of inactivation in this case was linearly dependent on the length of the side chain of 7a-d. These results show that the molecular yardstick approach tested here can provide useful information for modeling receptor proteins.

Chloroethylclonidine binds irreversibly to exposed cysteines in the fifth membrane-spanning domain of the human alpha2A-adrenergic receptor.

The alpha2-adrenergic receptors (alpha2-ARs) mediate signals to intracellular second messengers via guanine nucleotide binding proteins. Three human genes encoding alpha2-AR subtypes (alpha2A, alpha2B, alpha2C) have been cloned. Several chemical compounds display subtype differences in their binding and/or functional activity. Site-directed mutagenesis and molecular modeling are new tools with which to investigate the subtype selectivity of ligands. In this study, we introduce a new approach to mapping of the binding site crevice of the human alpha2A-AR. Based on a three-dimensional receptor model, we systematically mutated residues 197-201 and 204 in the fifth transmembrane domain of the human alpha2A-AR to cysteine. Chloroethylclonidine, an alkylating derivative of the alpha2-adrenergic agonist clonidine, binds irreversibly to alpha2A-ARs by forming a covalent bond with the sulfhydryl side chain of a cysteine residue exposed in the binding cavity, leading to inactivation of the receptor. Irreversible binding of chloroethylclonidine was used as a criterion for identifying introduced cysteine residues as being exposed in the binding cavity. The results supported a receptor model in which the fifth transmembrane domain is alpha-helical, with residues Val197, Ser200, Cys201, and Ser204 exposed in the binding pocket. Residues Ile198, Ser199, Ile202, and Gly203 face the lipid bilayer of the plasma membrane. This approach emerges as a powerful tool for structural characterization of the alpha2-ARs.

Alpha2-adrenoceptor regulation of adenylyl cyclase in CHO cells: dependence on receptor density, receptor subtype and current activity of adenylyl cyclase.

Chinese hamster ovary (CHO) cells stably transfected to express different densities of the human alpha2A-, alpha2B- and alpha2C-adrenoceptor subtypes, were used to characterize the regulation of adenylyl cyclase activity by alpha2-adrenoceptor agonists. In isolated cell membranes, activation of alpha2A- and alpha2C-adrenoceptors did not affect basal enzyme activity, but activation of alpha2B-adrenoceptors stimulated adenylyl cyclase activity. The extent of stimulation was dependent on the receptor density and was insensitive to pertussis toxin treatment. In the presence of 10 microM forskolin all three receptor subtypes mediated inhibition of adenylyl cyclase activity in a pertussis toxin-sensitive manner. In experiments performed with intact cells the same pattern could be seen: the basal production of cAMP was not affected when alpha2C-adrenoceptors were activated, but activated alpha2B-adrenoceptors mediated stimulation of cAMP production. In the presence of forskolin, both receptor subtypes mediated inhibition of cAMP production. Our results suggest that alpha2B-adrenoceptors are coupled to both Gi and Gs proteins. The signal transduction pathway to which the receptor is coupled is not dependent on receptor density, but its effect on adenylyl cyclase regulation is dependent on the current activity of adenylyl cyclase. The results also suggest that the alpha2A- and alpha2C-subtypes are preferentially coupled to Gi and transduce only inhibition of adenylyl cyclase activity in transfected CHO cells. At low densities of alpha2C-adrenoceptors, clonidine was a partial agonist, but in clones expressing high levels of alpha2C-adrenoceptors, clonidine acted as a full agonist by inhibiting cAMP accumulation with the same efficacy as (-)-noradrenaline. This demonstrates that receptor reserve can mask partial agonist activity.

Factors determining the specificity of signal transduction by guanine nucleotide-binding protein-coupled receptors. Integration of stimulatory and inhibitory input to the effector adenylyl cyclase.

To define the integration of multiple signals by different types of adenylyl cyclase (AC) within the cell, we altered the population of enzymes expressed in the cell and determined the subsequent processing of stimulatory and inhibitory input. DDT1-MF2 cells expressed AC VI-IX and were stably transfected with AC II, III, or IV. Enzyme expression was confirmed by RNA blot analysis and functional assays. Basal enzyme activity was only increased in AC II transfectants (6-fold). Maximum stimulation of enzyme activity was increased in each of the AC transfectants to varying extents. alpha2A/D-AR activation elicited enzyme type-specific responses. alpha2-AR activation inhibited the effect of isoproterenol in control transfectants, and this action was magnified in AC III transfectants. In AC II and AC IV transfectants, alpha2-AR activation initiated both positive (Gbetagamma) and negative signals (Gialpha) to the Gsalpha-stimulated enzyme, and both types of signals were blocked by cell pretreatment with pertussis toxin. The negative input to AC II from the alpha2-AR was blocked by protein kinase C activation in AC II transfectants, but it was the positive input to AC IV that was compromised by protein kinase C activation. These data indicate that the integration of multiple signals by adenylyl cyclases is a dynamic process depending upon the enzyme type and phosphorylation status.

Subtype specific recognition of human alpha2C2 adrenergic receptor using monoclonal antibodies against the third intracellular loop.

Monoclonal antibodies (Mabs) against human alpha2C2-adrenergic receptor (alpha2C2-AR) were raised in mice and characterized. Bacterially expressed fusion protein consisting a sequence from the putative third intracellular loop (amino acids 213-343) of human alpha2C2 and glutathione-S-transferase (GST) was used as antigen. Results from mass spectrometry of purified thrombin cleaved alpha2C2 polypeptide suggested that the epitope region would lie near the aminoterminal end of the 3rd intracellular loop of human alpha2C2-AR. Elevation of Mabs was detected with Western blotting from mouse blood samples. Three alpha2C2 specific cell clones were expanded to in vitro production in hollow fiber systems. The specificity of the Mabs was further determined by immunoprecipitation and immunocytochemistry. Scatchard analysis of thrombin digested, purified, Europium-labelled antigen (amino acids 213-343 of alpha2C2) revealed binding affinity constants of 0.4 x 10(9), 0.7 x 10(9) and 1.6 x 10(9) M(-1) and Kds of 2.6, 1.4 and 0.6 nM for the three Mabs 2B1, 3G3 and 7G1, respectively.

Molecular pharmacology of alpha 2-adrenoceptor subtypes.

alpha 2-adrenergic receptors mediate many of the physiological actions of the endogenous catecholamines adrenaline and noradrenaline, and are targets of several therapeutic agents. alpha 2-adrenoceptor agonists are currently used as antihypertensives and as veterinary sedative anaesthetics. They are also used in humans as adjuncts to anaesthesia, as spinal analgesics, and to treat opioid, nicotine and alcohol dependence and withdrawal. Three human alpha 2-adrenoceptor subtype genes have been cloned and designated alpha 2-C10, alpha 2-C4, and alpha 2-C2, according to their location on human chromosomes 10, 4 and 2. They correspond to the previously identified pharmacological receptor subtypes alpha 2A, alpha 2C and alpha 2B. The receptor proteins share only about 50% identity in their amino acid sequence, but some structurally and functionally important domains are very well conserved. The most obvious functionally important differences between the receptor subtypes are based on their different tissue distributions; e.g. the alpha 2A subtype appears to be an important modulator of noradrenergic neurotransmission in the brain. The three receptors bind most alpha 2-adrenergic drugs with similar affinities, but some compounds (e.g. oxymetazoline) are capable of discriminating between the subtypes. Clinically useful subtype selectivity cannot be achieved with currently available pharmaceutical agents. The second messenger pathways of the three receptors show many similarities, but small functional differences between the subtypes may turn out to have important pharmacological and clinical consequences. All alpha 2-adrenoceptors couple to the pertussis-toxin sensitive inhibitory G proteins Gi and G(o), but recent evidence indicates that also other G proteins may interact with alpha 2-adrenoceptors, including Gs and Gq/11. Inhibition of adenylyl cyclase activity, which results in decreased formation of cAMP, is an important consequence of alpha 2-adrenoceptor activation. Many of the physiological effects of alpha 2-adrenoceptor activation cannot, however, be explained by decreases in cAMP formation. Therefore, alternative mechanisms have been sought to account for the various effects of alpha 2-adrenoceptor activation on electrophysiologic, secretory and contractile cellular responses. Recent results obtained from studies on ion channel regulation point to the importance of calcium and potassium channels in the molecular pharmacology of alpha 2-adrenoceptors.

Use of a hollow fiber bioreactor for large-scale production of alpha 2-adrenoceptors in mammalian cells.

Gene cloning has revealed the existence of receptors, which are structurally similar but pharmacologically distinct. One recent example is the alpha 2-adrenergic receptor (alpha 2AR) family with three members. Preparation of membrane-embedded G-protein coupled receptor subtypes in pure form is practically impossible from natural sources and only recombinant techniques have provided possibilities to study these receptors in great detail. In this respect, both yeast and insect cell hosts have been applied successfully but no good mammalian alternative has been described for large-scale production. We describe in this report the use of S115 mouse mammary tumor cells as an effective host for large-scale production of alpha 2-adrenoceptors. These cells can be easily adapted to grow in a hollow fiber bioreactor, with up to 2.8 g of total cellular protein produced in one 0.8 m2 casette. We also show that each recombinant alpha 2-subtype exhibits their expected ligand binding properties, and suggest therefore that this system could be generally applicable to other eukaryotic plasma membrane proteins.

Similar ligand binding in recombinant human alpha 2 C2-adrenoceptors produced in mammalian, insect and yeast cells.

Ligand binding properties were investigated in recombinant human alpha 2C2-adrenoceptors expressed in three different host systems: Shionogi S115 mouse mammary tumour cells, Spodoptera frugiperda Sf9 insect cells and Saccharomyces cerevisiae yeast cells. The expected 43 kDa alpha 2C2 protein was visualized with immunoblotting using a polyclonal alpha 2C2-receptor antibody. [3H]Rauwolscine binding in cell homogenates or membranes (Bmax 3-11 pmol/mg protein; Kd approximately 5.5 nM) was inhibited by prazosin, oxymetazoline, RX821002, chlorpromazine and (-)-noradrenaline with and without the GTP-analogue Gpp(NH)p with similar Ki values in the different host systems. This indicates that alpha 2C2-adrenoceptors retain their binding characteristics irrespective of the host environment.

Coupling of human alpha 2-adrenoceptor subtypes to regulation of cAMP production in transfected S115 cells.

Stable S115 mouse mammary tumour cell lines, expressing separately alpha 2A-C10, alpha 2B-C2 and alpha 2C-C4 adrenoceptors were used to compare the receptor binding properties of alpha 2-adrenoceptor agonists with their potency in inhibiting cAMP production. All tested agonists detected high and low affinity binding sites in all three receptor subtypes. In the presence of the GTP analogue Gpp(NH)p (10 microM), all displacement curves were shifted to the right and were best modelled by one-site fits, suggesting that the receptor subtypes are coupled to G-proteins. The extent of the Gpp(NH)p-induced shift was greatest in the alpha 2A-C10 subtype, smaller in alpha 2C-C4, and minimal in alpha 2B-C2. All three receptor subtypes were also coupled to inhibition of forskolin-stimulated cAMP production through pertussis toxin-sensitive G-proteins. For the full agonists noradrenaline, UK 14,304, and dexmedetomidine, the maximal inhibitory effect on cAMP production was smaller in the alpha 2B-C2 subtype (35%) than in the alpha 2A-C10 and alpha 2C-C4 subtypes (50-70%). After treatment of cells expressing alpha 2B-C2 receptors with pertussis toxin, cAMP production was increased by up to 58% by alpha 2-adrenoceptor agonists. Similar stimulation of adenylyl cyclase activity could not be demonstrated at the other two receptor subtypes. In conclusion, these results demonstrate that (1) alpha 2-adrenoceptor agonists may be characterized by an agonist-type binding pattern in homogenates of transfected S115 cells, (2) all three alpha 2-adrenoceptor subtypes are coupled to inhibition of adenylyl cyclase in S115 cells through pertussis toxin-sensitive G-proteins, (3) the receptor-effector coupling in S115 cells is different among the subtypes so that the alpha 2A-C10 subtype is coupled with high efficacy but with low sensitivity, the alpha 2B-C2 subtype with low efficacy but high sensitivity, and the alpha 2C-C4 subtype with both high efficacy and high sensitivity, and (4) at least alpha 2B-C2 receptors may also be coupled to stimulation of adenylyl cyclase activity, presumably through Gs.

Use of recombinant human alpha 2-adrenoceptors to characterize subtype selectively of antagonist binding.

Cloning of the genes encoding three subtypes of human alpha 2-adrenoceptors allows the separate heterologous expression of each subtype. We have generated stably transfected Shionogi S115 mouse mammary tumour cell lines expressing the human alpha 2-adrenoceptor subtypes alpha 2-C10, alpha 2-C2, and alpha 2-C4 at densities of 0.2-7 pmol/mg total cellular protein. Binding of [3H]rauwolscine was inhibited by co-incubation of S115 cell homogenates with ten alpha 2-adrenoceptor antagonists and oxymetazoline, a partial agonist known to discriminate the receptor subtypes. Other useful agents for discrimination of subtypes were prazosin, chlorpromazine, phentolamine, and yohimbine. The most sensitive indices for differences between the three subtypes were the binding inhibition coefficient (Ki) ratios chlorpromazine/oxymetazoline (alpha 2-C10: 202; alpha 2-C2: 0.004; alpha 2-C4: 0.8), prazosin/oxymetazoline (430; 0.03; 0.5) and chlorpromazine/atipamezole (1612; 5.8; 77). Correlation analysis between our results for human-type receptors and published data for their rat alpha 2-adrenoceptor homologues demonstrated excellent general agreement, with some interspecies differences in the affinity of rauwolscine, phentolamine and oxymetazoline. The use of recombinant human receptors produced in stably transfected cell lines should facilitate the development of new, subtype-selective alpha 2-adrenoceptor ligands.

Comparison of four in situ hybridization methods, based on digoxigenin- and biotin-labelled probes, in detecting HPV DNA in male condylomata acuminata.

We have compared the efficacy of digoxigenin- and biotin-labelled probes in detecting HPV DNA by in situ hybridization on paraffin-embedded tissue sections of 57 male condyloma-suspect genital lesions. Each biopsy was hybridized with at least three of the following four methods: digoxigenin-labelled HPV DNA probes (Dig-HPV), biotinylated HPV-DNA probes (Bio-HPV), and two commercial methods (ViraType in situ and PathoGene), both based on biotinylated DNA probes. The hybridization products were visualized with colourigenic enzyme substrates. In most biopsies, the 4 methods gave equal results although cross-hybridization was most often found with the low-stringency ViraType method. Dig-HPV 6/11 probes gave positive results about twice as often as either of the commercial methods. No such difference, however, was found for HPV 16/18 probes. DNA of any type of HPV 6/11, 16/18 or 31/33/35 or 51 was detected in 28/43 (65%) of lesions showing condyloma acuminatum histology but in none of the 14 biopsies with no histological signs of HPV infection. In HPV-positive condylomata with no cellular atypia. HPV 6/11 was detected in 87% (13/15), and HPV 16/18 in 27% (4/15). In biopsies with cellular atypia, HPV types 6/11 were detected in 62% (8/13), HPV types 16/18 in 46% (6/13), and HPV types 31/33/35 or 51 in 50% (6/12). In about 50% of the biopsies where at least one hybridization method gave a positive result, either one of the commercial methods gave a negative result.(ABSTRACT TRUNCATED AT 250 WORDS)

Stable expression of recombinant human alpha 2-adrenoceptor subtypes in two mammalian cell lines: characterization with [3H]rauwolscine binding, inhibition of adenylate cyclase and RNase protection assay.

Cloning of the genes encoding distinct subtypes of human alpha 2-adrenergic receptors (alpha 2-AR) allows the separate recombinant expression of each individual subtype in heterologous systems. We report here the transfection, selection and preliminary pharmacological characterization of two mammalian cell lines, adherent Shionogi S115 mouse mammary tumour cells and human B-lymphoblastoid IBW4 cells growing in suspension, expressing the human alpha 2-AR subtypes alpha 2-C4 and alpha 2-C10 at densities of approx. 2 x 10(5) receptors/cell. Transfection of the subtype genes was verified using a specific RNase protection assay. Pharmacological characterization was carried out with [3H]rauwolscine binding, which was inhibited by oxymetazoline and prazosin in a subtype-selective manner. The sensitivity of (-)-noradrenaline binding to the GTP-analogue 5'-guanylylimidodiphosphate suggested that the receptors are coupled to G-proteins. This was verified in S115 cells by efficient inhibition of forskolin-stimulated cAMP production by the alpha 2-AR agonists, (-)-noradrenaline and clonidine. These cell lines thus appear to be suitable for pharmacological studies on receptor function and ligand binding.