Biomedical subjects

Sándor Benyhe

Publications and source records attributed to Sándor Benyhe.

16 recordsLinked to original sources

PubMed2007

Beta-methyl substitution of cyclohexylalanine in Dmt-Tic-Cha-Phe peptides results in highly potent delta opioid antagonists.

The opioid peptide TIPP (H-Tyr-Tic-Phe-Phe-OH, Tic:1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) was substituted with Dmt (2',6'-dimethyltyrosine) and a new unnatural amino acid, beta-MeCha (beta-methyl-cyclohexylalanine). This double substitution led to a new series of opioid peptides displaying subnanomolar delta antagonist activity and mu agonist or antagonist properties depending on the configuration of the beta-MeCha residue. The most promising analog, H-Dmt-Tic-(2S,3S)-beta-MeCha-Phe-OH was a very selective delta antagonist both in the mouse vas deferens (MVD) assay (Ke = 0.241 +/- 0.05 nM) and in radioligand binding assay (K i delta = 0.48 +/- 0.05 nM, K i mu/K i delta = 2800). The epimeric peptide H-Dmt-Tic-(2S,3R)-beta-MeCha-Phe-OH and the corresponding peptide amide turned out to be mixed partial mu agonist/delta antagonists in the guinea pig ileum and MVD assays. Our results constitute further examples of the influence of Dmt and beta-methyl substitution as well as C-terminal amidation on the potency, selectivity, and signal transduction properties of TIPP related peptides. Some of these compounds represent valuable pharmacological tools for opioid research.

Animals↗

PubMed2006

In vitro binding and functional studies of Ac-RYYRIK-ol and its derivatives, novel partial agonists of the nociceptin/orphanin F/Q receptor.

Following the discovery of nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) and its endogenous ligand, an extensive search has started to find selective agonists and antagonists targeting this novel receptor-ligand system due to their therapeutic potentials. By the help of the combinatorial chemistry a series of hexapeptides with a general formula of Ac-RYY-R/K-W/I-R/K-NH(2) having high NOP receptor affinity and selectivity were identified. On the basis of this information we developed a number of novel compounds. The detailed structure-activity studies on the partial agonist Ac-RYYRIK-NH(2) are reported in this communication. Besides the modifications on N- and C-terminal, Arg-Cit exchange was performed on the template structure. The novel hexapeptides were analyzed in radioligand binding, functional biochemical [(35)S]GTPgammaS binding assays by using membranes from rat brains and Chinese hamster ovary cells expressing human NOP receptor. The agonist/antagonist properties were also tested on in the mouse vas deferens bioassay. C-terminal modification yielded a high affinity, selective and potent NOP ligand (Ac-RYYRIK-ol) with a partial agonist property. Several analogs of this compound were synthesized. The presence of the positively charged arginine residue at the first position turned out to be crucial for the biological activity of the hexapeptide. The N-terminal modifications with various acyl groups (ClAc, pivaloyl, formyl, benzoyl, mesyl) decreased the affinity of the ligand towards the receptor and the intrinsic activity for stimulating the G-protein activation was also decreased. The structure-activity studies on the hexapeptide derivatives provided some basic information on the structural requirements for receptor binding and activation.

Animals↗

PubMed2006

In vitro and in vivo pharmacological characterization of the nociceptin/orphanin FQ receptor ligand Ac-RYYRIK-ol.

It was recently reported that the hexapeptide Ac-RYYRIK-ol binds with high affinity nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptors and competitively antagonizes N/OFQ actions in the mouse vas deferens assay. Here we further describe the in vitro and in vivo pharmacological features of this NOP receptor ligand. In mouse brain homogenate the degradation half life of Ac-RYYRIK-ol (2.48 min) was significantly higher than that of the parent compound Ac-RYYRIK-NH2 (1.20 min). In the electrically stimulated mouse vas deferens, Ac-RYYRIK-ol (10-1000 nM) competitively antagonized the inhibitory effect of N/OFQ (pA2=8.46), while in the isolated mouse colon the hexapeptide mimicked N/OFQ contractile effects thus behaving as a NOP receptor agonist (pEC50=9.09). This latter effect was no longer evident in colon tissues taken from mice knock out for the NOP receptor gene (NOP-/-). In vivo in mice, similarly to N/OFQ, Ac-RYYRIK-ol (dose range 0.001-1 nmol) produced: i) pronociceptive effects after intracerebroventricular (i.c.v.) administration and antinociceptive actions when given intrathecally (i.t.) in the tail withdrawal assay; ii) inhibition of locomotor activity and iii) stimulation of food intake after supraspinal administration. Finally, in the forced swimming test, Ac-RYYRIK-ol was inactive per se, but reversed the antidepressant-like effects elicited by the NOP receptor selective antagonist UFP-101 ([Nphe(1),Arg(14),Lys(15)]N/OFQ-NH2). Thus, in all these in vivo assays Ac-RYYRIK-ol mimicked the actions of N/OFQ showing however higher potency. In conclusion, Ac-RYYRIK-ol displayed a complex pharmacological profile which is likely due to the low efficacy agonist nature of this novel ligand of the NOP receptor. The high potency, selectivity of action, and in vivo effectiveness make Ac-RYYRIK-ol a useful pharmacological tool for future studies in the field of N/OFQ and its NOP receptor.

Animals↗

PubMed2006

Partial and full agonism in endomorphin derivatives: comparison by null and operational model.

The partial mu-opioid receptor pool inactivation strategy in isolated mouse vas deferens was used to determine partial agonism of endomorphins and their analogs (endomorphin-1-ol, 2',6'-dimethyltyrosine (Dmt)-endomorphin-1, endomorphin-2-ol and (D-Met2)-endomorphin-2) using morphine, normorphine, morphiceptin, (D-Ala2,MePhe4,Gly5-ol)-enkephalin (DAMGO) and its amide (DAMGA) as reference opioid agonists. Agonist affinities (KA) and efficacies were assessed both by the "null" and the "operational" method. The KA values determined by the two methods correlated significantly with each other and also with the displacing potencies against 3H-naloxone in the receptor binding assay in the presence of Na+. DAMGO and DAMGA were full agonist prototypes, morphine, endomorphin-1, endomorphin-1-ol, Dmt-endomorphin-1, endomorphin-2-ol and (D-Met2)-endomorphin-2 were found by both methods to be partial agonists whereas the parameters for normorphine, morphiceptin and endomorphin-2 were intermediate.

Animals↗

PubMed2006

Synthesis of enzymatically resistant nociceptin-related peptides containing a carbamic acid residue.

Nociceptin, a 17-amino acid peptide (FGGFTGARKSARKLANQ, N/OFQ), is the endogenous ligand of the nociceptin/orphanin FQ (NOP) receptor. This receptor-ligand system is involved in various physiological as well as pathophysiological mechanisms, but owing to the peptidic structure, it is rapidly degraded by enzymes. The enzymatic digestion of nociceptin involves mainly aminopeptidases and yields Noc(2-17)-OH and other smaller fragments. We aimed at increasing the enzymatic stability against aminopeptidases in the case of peptide Noc(1-13)-NH(2), which possesses the minimum sequence capable of interacting with the NOP receptor. Therefore we developed a new procedure for the synthesis of peptides with the carbamic acid residue [...-NH-CH(R)-CO-NH-CO-NH-CH(Q)-CO-.]. A set of four carbamic acid-nociceptin derivatives were produced. The carbamic acid residue was incorporated into the inner part of the peptides, building on solid phase, by using a suitable dipeptide fragment with carbamic acid residue produced by a simple and efficient three-step solution phase procedure. Enzymatic stability of carbamic acid peptides was studied in the presence of aminopeptidase M (AP-M) and in rat brain membrane homogenate. The receptor-binding properties were also studied by radioligand binding assay on crude rat brain membranes and the activity of the ligands were analyzed on isolated mouse vas deferens (MVD) tissues. We found that incorporation of the carbamic acid residue into the N-terminal part of nociceptin significantly increases the resistance against AP-M. We observed the decrease of binding affinities to the NOP receptor in case of the peptides modified in the N-terminal portion. Consequently, the incorporation of the carbamic acid residue into peptides can be proposed as a promising and reasonable tool for increasing enzymatic stability, where the native molecule is less sensitive for carbamic acid residue-related structural change.

Animals↗

PubMed2005

Synthesis and biological studies of nociceptin derivatives containing the DTPA chelating group for further labeling with therapeutic radionuclides.

Nociceptin is an endogenous anti-opiate heptadecapeptide primarily interacting with the nociceptin (NOP) receptor. This neuropeptide-receptor system is involved in pain regulation, tolerance to and dependence on opiates as well as many other physiological and pathophysiological events. The role and mechanisms of nociceptin in pathological conditions is not clearly known yet. In an attempt to have a radiopharmaceutical labeled either with 99mTc or (111)In, we incorporated diethylenetriaminepentaacetic acid (DTPA) as chelator into the structure of [Arg14,Lys15]nociceptin(1-17)-NH2 at the epsilon-amino group of Lys15. Such a radiopeptide may be useful in imaging for diagnostical purposes. Preparation of the peptide ligands was carried out by solid phase synthesis. Two peptides containing DTPA were obtained and purified. The products were [Arg14,Lys(DTPA)15]nociceptin(1-17)-NH2 and its cross-linked dimer on the basis of mass spectrometric analysis. In (115)In3+ binding experiments the conjugates exhibited preserved indium ion chelating properties, indicating the potential use of radiolabeled DTPA-nociceptin derivatives as radiopharmaceutical. Biological properties of these compounds were studied in rat brain membrane preparations by radioligand binding, functional biochemical [35S]GTPgammaS binding assays and mouse vas deferens (MVD) bioassay. Besides the similar in vitro binding characteristics to nociceptin receptor, both of the DTPA-chelated compounds were more potent and efficient than nociceptin in functional biochemical and mouse vas deferens bioassays. Our further aim is to radiolabel these compounds in order to get a radiopharmaceutical which can be used diagnostically.

Animals↗

PubMed2005

Terbutaline increases the cervical resistance of the pregnant rat in vitro.

Cervical ripening is a crucial process leading to delivery. Early dilation of the pregnant cervix can contribute to premature labour. The maturity of the cervix can be characterized by its resistance to mechanical stretching. Although a number of compounds are considered to increase cervical resistance (e.g., progesterone, nitric oxide synthase inhibitors and nonsteroidal anti-inflammatory drugs), none of them seem to be safe for clinical application. Other compounds, such as beta(2)-adrenergic receptor (beta(2)-AR) agonists, have been used for several decades to stop premature myometrium contractions, but their cervical action has never been investigated. The aim of this study was to detect the effects of the beta(2)-AR agonist terbutaline on nonpregnant and late-pregnant (day 18, 20, 21 or 22) cervices isolated from Sprague-Dawley rats. Cervical resistance was measured by means of a mechanical stretching test in vitro, the beta(2)-AR density was determined by Western blot analysis, the beta(2)-AR mRNA was determined by RT-PCR, while the G-protein activation following cervical beta(2)-AR stimulation with terbutaline was evaluated via a [(35)S]GTPgammaS binding assay. Terbutaline at 10(-6) M increased the cervical resistance of the late-pregnant samples in vitro from day 18 to day 22, but did not alter the resistance of the nonpregnant samples. This cervical resistance-increasing effect was concentration dependent and antagonized with propranolol on day 21. Terbutaline was ineffective on cervical samples when gradual stretching was omitted. RT-PCR and Western blot studies revealed increased beta(2)-AR mRNA and beta(2)-AR levels respectively on day 18 of pregnancy compared with the nonpregnant cervix, but no further changes were detected up to the end of pregnancy. The [(35)S]GTPgammaS binding assay demonstrated a decreased G-protein activation on the days of pregnancy investigated, but no activation was found in the nonpregnant samples. The degree of decrease in G-protein activation by terbutaline was in harmony with its cervical resistance-increasing action. On day 21, the G-protein activation-decreasing effect of terbutaline was antagonized with propranolol. We presume that the cervical resistance-increasing effect of terbutaline is a consequence of its G-protein activation-decreasing property via beta(2)-ARs, which finally leads to an increased muscle resistance against mechanical stretching. This action of terbutaline seems unique among the smooth muscles, and may open up a new perspective in the prevention of premature labour. Clinical experience indicates that beta(2)-AR agonists will not be sufficient to stop the overall process, but their combination with more potent inhibitors of uterine contractions may be of clinical benefit.

Adrenergic beta-Agonists↗

PubMed2005

Opioid receptor binding characteristics and structure-activity studies of novel tetrapeptides in the TIPP (Tyr-Tic-Phe-Phe) series.

The development of the prototype synthetic delta-opioid receptor antagonist peptides TIPP [(H-Tyr-Tic-Phe- Phe-OH); Tic: tetrahydroisoquinoline-3-carboxylic acid] and TIPPpsi (H-Tyr-psiTic-Phe-Phe-OH) by Schiller and coworkers was followed by extensive structure-activity relationship studies, leading to the emergence of numerous analogs that are of pharmacological interest. Eight novel diastereomeric compounds in this peptide family were designed, prepared, and tested biologically to gain structure-activity relationship information. The new multisubstituted tetrapeptide analogs contain both a 2',6'-dimethyltyrosine residue at the N-terminus and beta-methyl-cyclohexylalanine at the third position as replacements for the original first tyrosine and the third phenylalanine, respectively. These derivatives wear either free acidic (-COOH) or amidated (-CONH2) C-terminal. The potency and delta- versus mu-opioid receptor selectivity were evaluated by in vitro radioreceptor-binding assays, while the intrinsic G-protein-activating efficacy of these analogs was tested in [35S]GTPgammaS-binding assays using rat brain membranes or Chinese hamster ovary cells stably expressing mu- or delta-opioid receptors. The analogs showed delta-antagonist selectivity with differences regarding their isomeric forms, and these analogs containing a C-terminal carboxamide group displayed a mixed mu-agonist/delta-antagonist profile, thus they are expected to be safer analgesics with a low propensity to produce tolerance and physical dependence. These results constitute further examples of the influence of beta-methyl substitution and C-terminal amidation on potency, selectivity, and signal transduction properties of TIPP-related peptides as well as they represent valuable pharmacological tools for opioid research.

Analgesics, Opioid↗

PubMed2005

Pregnancy-induced decrease in the relaxant effect of terbutaline in the late-pregnant rat myometrium: role of G-protein activation and progesterone.

The effectiveness of beta2-agonists in preterm delivery is reduced by several factors. The aim of this study was to determine the influence of late pregnancy in the uterus-relaxing effect of terbutaline in the rat in vitro. Rat uterine tissues from late pregnancy (days 15, 18, 20 and 22) were used. In vitro electrical field-stimulation (EFS) was used to evoke contractions. The radioligand-binding technique, reverse transcription-polymerase chain reaction and radioimmunoassay technique were used to determine the beta-adrenergic receptor density and mRNA level and the plasma sex hormone level, respectively. The activated G-protein level of the beta-adrenergic receptors was investigated by a radiolabelled GTP binding assay.EFS-induced contractions were inhibited by terbutaline. This effect decreased towards term with respect to both the EC50 and maximal inhibition values. A drop in plasma progesterone level was also detected. Binding studies revealed an increase in beta-adrenergic receptor number on the last day of pregnancy, which correlated with the change in receptor mRNA level. The G-protein-activating effect of terbutaline decreased continuously between days 15 and 20. Surprisingly, terbutaline decreased the G-protein activation to below the basal level on day 22. However, progesterone pretreatment set back the uterine action of terbutaline, increased the density of the beta2-adrenergic receptors and their mRNA level and increased the G-protein-activating property of terbutaline. These data provide evidence of a pregnancy-induced decrease in activated G-protein level after beta2-agonist stimulation. The decrease in plasma progesterone level has a crucial role in this process. The effects of beta2-adrenergic receptor agonists in tocolytic therapy may possibly be potentiated with progesterone.

Animals↗

PubMed2004

Nociceptin antagonism: probing the receptor by N-acetyl oligopeptides.

In search for effective antagonist structures for the nociceptin (NOP) receptor, a number of N-acylated oligopeptides, including N-acyl tetra- and pentapeptides selective for the kappa-opioid receptor, as well as N-acyl hexapeptides bearing the Ac-Arg-Tyr-Tyr-Arg-Ile-Lys (Ac-RYYRIK) core sequence originally isolated from combinatorial chemical libraries, were synthesized and studied in radioreceptor binding assays, [(35)S]GTPgammaS functional tests and in mouse vas deferens (MVD) bioassays. The properties of the novel antagonist candidates were compared to known antagonists. A new antagonist structure with a reduced, primer alcohol C-terminus, Ac-Arg-Tyr-Tyr-Arg-Ile-lysinol (Ac-RYYRIK-ol) was described in the mouse vas deferens tests, showing an equilibrium inhibitory constant value (K(e)) of 2.44 nM, and no agonist effect at 10 microM ligand concentration. Schild-analysis indicated a clearly competitive interaction at the NOP receptor, whereas the peptide did not affect the action of the delta-opioid receptor agonist [D-Ala(2),D-Leu(5)]enkephalin. Ac-RYYRIK-ol also exhibited a high affinity in [(3)H]nociceptin-NH(2) binding competition assays using rat brain membranes. Agonist-induced G-protein activation via NOP receptors was studied in [(35)S]GTPgammaS binding stimulation assays by the use of both native brain tissue preparations and membranes from cultured CHO cells expressing recombinant nociceptin receptors. Ac-RYYRIK-ol displayed only weak intrinsic agonist activity, whereas it effectively inhibited the stimulation generated by nociceptin. The results support the high potency and antagonist nature of Ac-RYYRIK-ol and reveal important roles for both the N- and the C-terminal region of the molecule.

Animals↗

PubMed2004

Pharmacological and functional biochemical properties of D-Ala2-D-Nle5-enkephalin-Arg-Phe.

Tyr-D-Ala-Gly-Phe-D-Nle-Arg-Phe (DADN) a synthetic analogue of the endogenous Met-enkephalin-Arg-Phe (Tyr-Gly-Gly-Phe-Met-Arg-Phe; MERF), was investigated in radioligand binding assays, [(35)S]GTPgammaS stimulation experiments as well as in in vivo algesiometric tests. Binding properties of [(3)H]DADN were measured in crude membrane fractions of rat spinal cord tissues and in homogenates of Chinese hamster ovary (CHO) cells selectively expressing delta-, kappa-or micro-opioid receptors. The highest affinity for [(3)H]DADN binding was observed in membranes from CHO cells transfected with micro-opioid receptors confirming the micro-selectivity of the peptide. Unlabeled DADN was also investigated in functional biochemical experiments by measuring opioid receptor-mediated G-protein activation in rat brain membrane fractions. The peptide stimulated the activity of the regulatory G-proteins in a concentration dependent manner, and the stimulation was efficiently inhibited in the presence of micro-receptor specific antagonist ligands further supporting the selectivity profile of DADN. Intrathecally administered DADN produced a dose-related, naloxone-reversible antinociception in rat hot water tail-flick tests. Among the selective opioid antagonists tested, the delta-selective naltrindole (NTI) and the kappa-specific norbinaltorphimine (norBNI) showed only slight blocking effects compared with naloxone. The results obtained in the in vitro agonist-stimulated [(35)S]GTPgammaS binding assays are in good agreement with the opioid agonist effect seen in the in vivo pain test.

Animals↗

PubMed2004

Non-opioid actions of opioid peptides.

Beside the well known actions of opioid peptides on mu-, delta- and kappa-opioid receptors, increasing amount of pharmacological and biochemical evidence has recently been published about non-opioid actions of various opioid peptides. These effects are not abolished by naloxone treatments. Such non-opioid effects are observed both in nervous tissues and in the cellular elements of the immune system. Peptides exhibiting non-opioid effects include beta-endorphin, dynorphin A, nociceptin/OFQ, endomorphins, hemorphins and a number of Proenkephalin A derived peptides, such as Met-enkephalin, Met-enkephalin-Arg-Phe (MERF) and bovine adrenal medullary peptide (BAM22). Non-opioid actions are exerted through different neuronal receptors, e.g., dynorphin hyperalgesia through NMDA receptor, Met-enkephalin induced regulation of cell growth through zeta receptors, pain modulation by nociceptin through ORL-1 or NOP receptors, while BAM22 acts through sensory neuron specific G protein-coupled receptors (SNSR). We have investigated Met-enkephalin-Arg-Phe (MERF) and its analogues by the means of direct and indirect radioligand binding assays. It has been found that in addition to kappa(2) and delta-opioid receptors, MERF can act also through sigma(2)- or probably via FMRF-NH(2) receptors in rat cerebellum. A role of functionally assembling heterodimer receptors in mediating the non-conventional actions of these peptide ligands can not be excluded as well.

Animals↗

PubMed2004

Structure-activity study on the Phe side chain arrangement of endomorphins using conformationally constrained analogues.

Endomorphins-1 and -2 were substituted with all the beta-MePhe stereoisomers in their Phe residues to generate a conformationally constrained peptide set. This series of molecules was subjected to biological assays, and for beta-MePhe(4)-endomorphins-2, a conformational analysis was performed. Incorporation of (2S,3S)-beta-MePhe(4) resulted in the most potent analogues of both endomorphins with enhanced enzymatic stability. Their micro opioid affinities were 4-times higher than the parent peptides, they stimulated [(35)S]GTPgammaS binding, and they were found to be full agonists. NMR experiments revealed that C-terminal (2S,3S)-beta-MePhe in endomorphin-2 strongly favored the gauche (-) spatial orientation which implies the presence of the chi(1) = -60 degrees rotamer of Phe(4) in the binding conformer of endomorphins. Our results emphasize that the appropriate orientation of the C-terminal aromatic side chain of endomorphins is substantial for binding to the micro opioid receptor.

Animals↗

PubMed2003

Synthesis and binding characteristics of a novel enkephalin analogue, [3H]Tyr-D-Ala-Gly-Phe-D-Nle-Arg-Phe.

The endogenous opioid heptapeptide (Tyr-Gly-Gly-Phe-Met-Arg-Phe; MERF) has been shown to interact with multiple opioid as well as non-opioid sites in mammalian brain membranes. To increase the stability and bioavailability of MERF, new synthetic derivatives with D-amino acid substitutions were prepared and studied. One of the new compounds in this series, Tyr-D-Ala-Gly-Phe-D-Nle-Arg-Phe (DADN), had only moderate affinity in competing with [3H]MERF, whereas it displayed the highest potency in producing antinociception following intrathecal administration. DADN was radiolabeled with 41Ci/mmol specific activity. Specific binding of [3H]DADN was saturable, stereoselective and of high affinity. Chemical stability, increased micro-receptor selectivity, and hydrophobicity of the peptide all contribute to the effectiveness observed in biochemical and pharmacological studies.

Amino Acid Sequence↗

PubMed2002

Biochemical characterisation of newly developed beta-etorphine and beta-dihydroetorphine derivatives.

The highly potent synthetic narcotic compound etorphine is known to cause strong analgesia, catatonia and blockade of conditioned reflexes in laboratory animals and is widely used for the immobilisation of game animals. In this study, a number of new structural analogues of etorphine, including C18-beta-structures, 3-O-methylether derivatives and saturated C7-C8 dihydro-compounds, were synthesised and examined in in vitro ligand binding experiments. Opiate receptor-mediated activation of G-proteins by these derivatives was also investigated using the [35S]GTPgammaS binding assay. The receptor binding affinity constant and G-protein stimulatory potency of the novel beta-etorphins were compared with those of the corresponding C18-alpha-derivatives. In rat brain membrane preparations, all the compounds tested displayed high affinity (K(i)'s ranging 0.4-22 nM) using [3H]naloxone in competition assays. The alpha-etorphines had somewhat higher affinity in comparison with the beta-structures. Methylether derivatives were consistently weaker than the corresponding phenolic compounds. Dihydroetorphine and beta-dihydroetorphine, which have a partially saturated ring structure, showed as good potency in the binding assays as did etorphine and beta-etorphine with C7-C8 double bonds. The etorphine derivatives were potent but naloxone-reversible activators of G-proteins in the [35S]GTPgammaS functional tests. It was also found that the C3 phenolic group is favourable for G-protein activation. On the basis of our experimental results, neither the configuration of C18 nor the saturation of the C7-C8 double bond appears to play a critical role in the biological activity of etorphines.

Animals↗

PubMedDate not supplied

Effect of alpha-adrenoceptor subtype-selective inverse agonists on non-pregnant and late-pregnant cervical resistance in vitro in the rat.

1. The aim of the present study was to compare and elucidate the effects of alpha(1)-adrenoceptor (alpha(1)-AR) subtype-selective inverse agonists on non-pregnant and late-pregnant rat cervical tone. 2. Cervical resistance was investigated in in vitro stretching tests in the absence or presence of alpha(1)-AR subtype-selective inverse agonists (WB 4101, AH 11110A and BMY 7378; all at 10(-6) mol/L), whereas the mRNA levels and density of the alpha(1)-AR subtypes and the G-protein-activating effects of the inverse agonists were determined by reverse transcription-polymerase chain reaction, western blot and [(35)S]-GTPgammaS binding techniques, respectively. 3. The inverse agonists did not cause any change in resistance in non-pregnant and 18-day-pregnant samples. WB 4101 increased cervical resistance from Day 20, whereas AH 11110A had no effect and BMY 7378 exhibited such an action only on Day 21. Phenylephrine (10(-4) mol/L) had no effect on cervical resistance on Day 22. The mRNA levels and density of all alpha(1)-AR subtypes were increased on Day 18, but no further changes were observed after that. The [(35)S]-GTPgammaS binding studies revealed increased G-protein activation of alpha(1A)-AR and a moderate G-protein activation of alpha(1B)- and alpha(1D)-AR. The effect of WB 4101 to increase [(35)S]-GTPgammaS binding was blocked by pertussis toxin (50 ng/mL). Phenylephrine caused a slight and significant decrease in the amount of activated G-protein on Day 22. 4. The effects of inverse agonists on the alpha(1A)-AR can enhance cervical resistance in the late-pregnant rat in vitro. This action is mediated, at least in part, by a pertussis toxin-sensitive G(i)-protein. This effect of the alpha(1A)-AR inverse agonist WB 4101 may offer a new therapeutic target in the prevention of premature labour.

Adrenergic alpha-Agonists↗