ß-Blockers have differential effects on the murine asthma phenotype.

BACKGROUND AND PURPOSE: Our previous studies have shown the ß2 -adrenoceptor and its endogenous ligand, adrenaline, are required for development of the asthma phenotype in murine asthma models. Chronic administration of some, but not other, ß-blockers attenuated the asthma phenotype and led us to hypothesize that biased signalling was the basis of their differential effects, experimentally and clinically. EXPERIMENTAL APPROACH: We used mice with no detectable systemic adrenaline (PNMT(-/-) ) and wild-type (WT) mice to study the effects of four ß-blockers, alprenolol, carvedilol, propranolol and nadolol, in an ovalbumin sensitization and challenge (Ova S/C) murine model of asthma. The parameters measured were inflammatory cell infiltration, mucous metaplasia and airway hyperresponsiveness. To interpret the pharmacological action of these ligands quantitatively, we conducted computer simulations of three-state models of receptor activation. KEY RESULTS: Ova S/C PNMT(-/-) mice do not develop an asthma phenotype. Here, we showed that administration of alprenolol, carvedilol or propranolol in the absence of interference from adrenaline using Ova S/C PNMT(-/-) mice resulted in the development of an asthma phenotype, whereas nadolol had no effect. Ova S/C WT mice did develop an asthma phenotype, and administration of alprenolol, propranolol and carvedilol had no effect on the asthma phenotype. However, nadolol prevented development of the asthma phenotype in Ova S/C WT mice. Computer simulations of these four ligands were consistent with the isolated three-state receptor model. CONCLUSION AND IMPLICATIONS: ß-Blockers have different effects on the murine asthma phenotype that correlate with reported differences in activation or inhibition of downstream ß2 -adrenoceptor signalling pathways.

New perspectives regarding ß(2) -adrenoceptor ligands in the treatment of asthma.

In the last two decades several significant changes have been proposed in the receptor theory that describes how ligands can interact with G protein-coupled receptors (GPCRs). Here we briefly summarize the evolution of receptor theory and detail recent prominent advances. These include: (i) the existence of spontaneously active GPCRs that are capable of signalling even though they are unoccupied by any ligand; (ii) the discovery of ligands that can inactivate these spontaneously active receptors; (iii) the notion that a ligand may simultaneously activate more than one GPCR signalling pathway; and (iv) the notion that certain ligands may be able to preferentially direct receptor signalling to a specific pathway. Because the data supporting these receptor theory ideas are derived primarily from studies using artificial expression systems, the physiological relevance of these new paradigms remains in question. As a potential example of how these new perspectives in receptor theory relate to drug actions and clinical outcomes, we discuss their relevance to the recent controversy regarding the chronic use of ß(2) -adrenoceptor agonists in the treatment of asthma.

Predictors of seropositivity to herpes simplex virus type 2 in women.

Five hundred and twenty consecutive women newly attending a genitourinary medicine clinic who participated in a study of sexual behaviour were also tested for type-specific antibody to herpes simplex virus type 2; 135 (26%) were seropositive, of whom only 29 (21.5%) had had clinical evidence of genital herpes. Seropositive women were much more likely to have a past history of genital herpes (odds ratio [OR] 173). They were also more likely to be black non-UK born (OR 14), aged 30 years or over (OR 6), to have had 6-20 sexual partners (OR 3-4), especially from abroad (OR 12), to be unemployed (OR 6) or blue collar workers (OR 4), to have smoked cigarettes (OR 2) and to have practised peno-anal penetration (OR 5). Disease predictors included a past history of pelvic inflammatory disease (OR 63) and bacterial vaginosis (OR 3). Unexpected predictors were only one sexual partner (OR 5) and no non-regular partners (OR 5). Commencing intercourse before 16 years of age showed a protective effect (OR 0.2) and so did use of oral contraception (OR 0.5). Our findings show that infection with HSV-2 is associated with a wider range of morbidity and also emphasize the role of male sexual partner selection in the transmission of infection.

Is paradoxical pharmacology a strategy worth pursuing?

The first rule of medicine is, 'do no harm'. Perhaps accepting this precept has produced a logic for disease treatment where our primary purpose is limited to only assisting or helping to correct a malfunctioning system. Can we use drugs that, according to traditional views, would be considered to increase stress on the system in the short term, to actually treat and cure disease in the long term? Is it possible to exacerbate disease for a longer-term gain? Although there are several examples of where this strategy has appeared to work, a systematic testing of the hypothesis has not occurred and, for the majority of diseases, this hypothesis has never been tested.

Altered baroreflex responses in alpha7 deficient mice.

The autonomic nervous system controls and coordinates several cardiovascular functions, including heart rate, arterial pressure, blood flow and vasomotor tone. Neuronal nicotinic acetylcholine receptors (nAChRs) are the interface between the nervous system and the cardiovascular system, but it is not known which nAChR subtypes regulate autonomic function in vivo. Nicotinic AChRs containing the alpha7 subunit are a candidate subtype in autonomic ganglia. Stimulation of these nAChRs can increase neurotransmitter release via presynaptic mechanisms, as well as mediate fast synaptic transmission via postsynaptic mechanisms. To investigate the role of the alpha7 nAChR subunit in cardiac autonomic function, we measured baroreflex-mediated responses in alpha7 null mice. Here we show that the alpha7 null mice have impaired sympathetic responses to vasodilatation, as sodium nitroprusside infusion triggered a 48% heart rate increase in wild type mice but only a 21% increase in the alpha7 nulls (P < 0.001). The mutant mice developed supersensitivity to adrenergic agonists, although norepinephrine release from sympathetic nerve terminals could be elicited through mechanisms alternative to nAChR stimulation. Baroreflex-mediated parasympathetic responses were normal in alpha7 null mice. The decreased baroreflex-mediated tachycardia in alpha7 mutant mice indicates that alpha7-containing nAChRs participate in the autonomic reflex that maintains blood pressure homeostasis. The alpha7 mutant mice may serve as a model of baroreflex impairment arising from autonomic dysfunction.

Sexual behaviour and sexually transmitted infection among African and Caribbean men in London.

We studied 180 black heterosexual men of whom 133 (74%) were Caribbean and 47 (26%) African. Seventy-three per cent of Caribbeans and 27% of Africans were UK born. We found no difference in age, but more Africans were married (30% cf 10%; P=0.002) and students (26% cf 10%; P=0.00008). More Caribbeans smoked 1-10 cigarettes a day (42% cf 22%; P=0.02) and more drank alcohol (89% cf 74%; P=0.002). Sixty-nine per cent of Caribbeans reported intercourse before the age of 17 compared with 48% of Africans (P=0.004), but there was no difference in the numbers of sexual partners, either in the previous year or in total. Twenty-four (18%) of the 133 Caribbeans had gonorrhoea compared with one (2%) of the 47 Africans (P=0.001). Multivariate analysis showed that coitarche under 16 years of age (odds ratio (OR) 50) and gonococcal and/or chlamydial infection (OR 12.5) were independently associated with Caribbeans. Within this group, gonorrhoea was found more often in teenagers (OR 9.5) who had commenced intercourse before the age of 16 (OR 3.3) and chlamydial infection in those with multiple partners (OR 24). New problem-orientated approaches are needed to eradicate these curable infections which facilitate infection with HIV.

Treatment with inverse agonists enhances baseline atrial contractility in transgenic mice with chronic beta2-adrenoceptor activation.

In this study, we investigate whether chronic treatment with beta-adrenoceptor (betaAR) ligands with inverse agonist activity enhances myocardial beta2AR-mediated atrial tension more than neutral antagonists in transgenic mice (TG35). These mice exhibit chronic adrenoceptor activation because they possess a greater number of constitutively active receptors than wild type mice due to cardiac-specific overexpression of human betaARs. TG35 and wild type mice were chronically treated for 90 h with three inverse agonists, ICI-118,551, propranolol, and carvedilol, and one neutral antagonist, alprenolol. After 96 h, we compared the basal and isoprenaline-stimulated (10 microM) increase in atrial tension in treated or untreated TG35 mice and wild type mice. In parallel, to determine the effect of chronic betaAR ligand treatment on the amounts of G protein receptor kinase-2 (GRK-2) and G proteins, we performed Western blotting on myocardial cytosolic and membrane proteins. Atria from the TG35 mice treated with inverse agonists showed increases in the baseline tension compared to those from alprenolol/vehicle-treated mice. ICI-118,551 and propranolol treatment restored the elevated myocardial G-inhibitory protein (Gialpha) levels to that of wild type. Also, treatment with inverse agonists upregulated G-stimulatory protein (Gsalpha) levels and GRK2 above those levels in vehicle-treated TG35 or wild type mice. The increased baseline atrial tension was reversed by the addition of ICI-118,551. Overall, our data suggests that inverse agonists enhance baseline atrial tension more than neutral antagonists. Based on this, we propose that upregulation of the active conformation of the beta2ARs, Gsalpha protein and restoration of Gialpha as three possible mechanisms to explain this enhanced receptor activity. Therefore, the favourable effects of some ligands used in pathological conditions involving chronic adrenoceptor activation may be due to the inverse agonist activity of the ligand.

The elusive nature of intrinsic efficacy.

In the discipline of pharmacology, drugs (ligands) are used as tools to elucidate the processes of biological systems. Because of this, pharmacologists strive to delineate all characteristics of drugs. Decades of research have resulted in the proposal that ligands possess two properties that are intrinsic to the ligand and are invariant of the system in which their effects are investigated. These properties are affinity (the capacity of a drug to bind to a receptor) and intrinsic efficacy (the capacity of a drug to activate or inactivate a receptor). Although affinity is a relatively easy parameter to measure with a variety of techniques, ways of quantifying intrinsic efficacy have remained elusive ever since its inception. Furthermore, recent evidence suggests that intrinsic efficacy might not be a single, ligand-dependent parameter but that agonists might have multiple intrinsic efficacies. William Clarke and Richard Bond discuss several reasons why the claim that intrinsic efficacy is a ligand-dependent parameter should be questioned, and the possible impact of these findings.

Racial origin, sexual behaviour, and genital infection among heterosexual men attending a genitourinary medicine clinic in London (1993-4).

OBJECTIVES: To compare variables of sexual behaviour and incidence of genital infections among heterosexual men of different racial origins. DESIGN: A prospective cross sectional study of sexual behaviour reported by a standardised self administered questionnaire in new patients who presented for screening and diagnosis. SETTING: A genitourinary medicine clinic in west London. SUBJECTS: 1212 consecutive heterosexual men newly attending in 1993-4. MAIN OUTCOME MEASURES: Variables relating to sociodemographic status, sexual behaviour, condom use, sexually transmitted diseases, and other genital infections stratified by racial origin. RESULTS: There were 941 evaluable heterosexual men of whom the majority were white (79%) and 17% were black. The black men comprised more teenagers (11% cf 2%; p < 0.00001), were more likely to be unemployed (26% cf 12%; p < 0.00001), to have commenced intercourse much earlier (45% cf 22% before aged 16: p < 0.0001), and to have had intercourse with an African woman (14% cf 6%; p < 0.001). Both fellatio (64% cf 96%; p < 0.00001) and cunnilingus (40% cf 92%; p < 0.00001) were practised less frequently by the black men and so too was anal intercourse (11% cf 27%; p < 0.00001). Similar proportions from both groups were non-smokers (53% cf 57%), but a significantly higher proportion of the black men did not drink alcohol (13% cf 5%; p < 0.001). Gonorrhoea (15% cf 1%; p < 0.00001), chlamydial infection (17% cf 8%; p < 0.001), and non-gonococcal urethritis (37% cf 24%; p = 0.001) were diagnosed more frequently in the black men. These findings remained significant after logistic regression and are therefore independently associated with black race. However, there was no significant difference in numbers of sexual partners in the preceding year (median 2), nor in condom use with regular and non-regular partners. The Asian men had commenced intercourse later (mean 19.1 years) than both the black men (mean 15.9 years) and the white men (mean 17.3 years). CONCLUSIONS: Compared with white men, black men attending a genitourinary medicine clinic were much more likely to be unemployed, to have commenced intercourse earlier and to have urethral infection. They were much less likely to practice fellatio, cunnilingus, or anal intercourse. However, there was no difference between the two racial groups in respect of numbers of sexual partners and condom use.

Racial origin, sexual lifestyle, and genital infection among women attending a genitourinary medicine clinic in London (1992).

OBJECTIVES: To compare variables of sexual behaviour and incidence of genital infections among women of different racial origins and lifestyles. DESIGN: A prospective cross sectional study of sexual behaviour reported by a standardised self administered questionnaire in new patients who presented for screening and diagnosis. SETTING: A genitourinary medicine clinic in west London. SUBJECTS: 1084 consecutive women newly attending in 1992. MAIN OUTCOME MEASURES: Variables relating to sociodemographic status, sexual lifestyle, condom use, sexually transmitted diseases, and other genital infections stratified by racial origin. RESULTS: There were 948 evaluable women, of whom 932 (98.3%) were heterosexual and 16 (1.7%) were lesbian. Previous heterosexual intercourse was reported by 69% of lesbian women and their most frequent diagnosis was bacterial vaginosis (38%). The majority of heterosexual women were white (78%) and 16% were black. The black women were more likely to be teenagers (18% cf 8%; p = 0.0004) or students (28% cf 15%; p = 0.0008), and to have had an earlier coitarche (48% cf 38% before aged 17; p < 0.004). They also had a higher proportion of pregnancies (58% cf 38%; p < 0.00001) and births (38% cf 20%; p < 0.00001). The white women showed significantly more sexual partners during the preceding year (p = 0.004) and in total (p < 0.00001) and more reported non-regular partners (48% cf 35%; p = 0.004) with whom they were more likely to use condoms (p = 0.009). However, the black women were more likely to have gonorrhoea (7% cf 2% p < 0.0003), chlamydial infection (12% cf 5% p < 0.002), trichomoniasis (10% cf 2% p < 0.00001), or to sexual contacts of men with non-gonococcal urethritis (19% cf 12% p < 0.02). They were less likely to have genital warts (3% cf 12% p = 0.002). Logistic regression showed that all these variables were independently associated with the black women. The Asian women (2%), none of whom had a sexually transmitted disease, had commenced intercourse later (mean 19.7 years) than both black women (mean 16.8 years) and white women (mean 17.6 years). CONCLUSIONS: Sexual intercourse commenced approximately 1 year earlier in the black women, who were more likely to have become pregnant, had children, and to have acquired a bacterial sexually transmitted infection than were the white women.

Heterosexual behaviour, risk factors and sexually transmitted infections among self-classified homosexual and bisexual men.

Our study of men presenting at a genitourinary medicine clinic shows that self-classification into homosexual or bisexual does not accurately define behaviour. We found that 8.5% of self-defined homosexual men had had heterosexual intercourse in the past year and that 26% of self-defined bisexual men had not. Overall, 19% of homosexual/bisexual men reported vaginal intercourse in the past year and a further 42% in their lifetime. Compared with heterosexual men attending our clinic, the practising bisexual men were significantly more likely to come from a white ethnic group (P < 0.003) and to use condoms invariably with regular female partners (P = 0.0001). There was no significant difference in consent for HIV testing between homosexual (43%), practising bisexual (49%) and heterosexual (42%) men despite significantly different perceptions of risk. None of the practising bisexual men was seropositive for HIV infection (P = 0.06) or for syphilis (P = 0.02), or had chlamydial infection, which was found infrequently among homosexual men in general (P = 0.00001). HIV infection found in 19.4% of the exclusively homosexual men was associated with more frequent alcohol consumption (P=0.06).

An efficacy-dependent effect of cardiac overexpression of beta2-adrenoceptor on ligand affinity in transgenic mice.

In previous studies, it was shown that the overexpression of beta2-adrenoceptor (beta2AR) in the hearts of transgenic mice (Tg) leads to agonist-independent activation of adenylate cyclase and enhanced myocardial function. Here, we measured the physical coupling of beta2AR and Gs by evaluating the coimmunoprecipitation of beta2AR and Gs and the ligand binding properties of beta2AR in the hearts of Tg mice to investigate the details of the interaction among ligand, receptor, and G protein. The following results were obtained: (i) coimmunoprecipitation of beta2AR and Gs was increased in the absence of agonist in Tg mice compared with the control animals. This demonstrates directly the increased interaction between unliganded beta2AR and Gs, which is consistent with increased background cAMP production and cardiac function in the hearts of Tg mice. (ii) Guanosine-5'-(beta,gamma-imido)triphosphate abolished the association of beta2AR/Gs in the immunoprecipitate. (iii) The affinities for ligands that show agonist (isoproterenol, clenbuterol, and dobutamine), neutral antagonist (alprenolol and timolol), and negative antagonist (propranolol and ICI 118551) activities in this experimental system were increased, not changed and decreased, respectively, in Tg mice compared with the controls. (iv) This efficacy-dependent alteration in ligand affinities was still observed in the presence of a guanosine-5'-(beta,gamma-imido)triphosphate concentration that abolishes beta2AR/Gs coupling. This suggests that the altered beta2AR binding affinities in Tg mice are not due to the increased interaction between beta2AR and Gs. These data cannot be explained by using ternary, quinternary, two-state extended ternary, or cubic ternary complex models. We therefore discuss the results using a "two-state polymerization model" that includes an isomerization step for the conversion of receptor between an inactive and an active form (denoted as R and R*, respectively) and a polymerization of the active state (R*n). The simplest form of this model (i.e., noncooperative dimerization of the receptor) is found to be consistent with the experimental data.

Do recent operational studies indicate that a single state model is no longer applicable to G protein-coupled receptors?

Recent evidence suggests that unliganded G protein-coupled receptors exist in at least two states, an inactive conformation and an active conformation possessing affinity for the G protein even in the absence of agonist. The data accumulated so far for wild-type receptors imply that this is true for receptors for several hormones and receptor subtypes, and theoretically for all G protein-coupled receptors. The data now consist of studies implicating not only spontaneous receptor-G protein coupling, but also effector mechanisms and, in the case of transgenic mice over-expressing the human beta 2-adrenoceptor, physiologic responses at the level of the isolated tissue and in vivo. Furthermore, there appear to be ligands (inverse agonists) that can decrease the level of the constitutively active conformation of the receptor, and neutral antagonists can not only block classical agonist responses, but also inhibit the response of inverse agonists.

Ligand-induced overexpression of a constitutively active beta2-adrenergic receptor: pharmacological creation of a phenotype in transgenic mice.

Transgenic overexpression (40- to 100-fold) of the wild-type human beta2-adrenergic receptor in the hearts of mice leads to a marked increase in cardiac contractility, which is apparently due to the low level of spontaneous (i.e., agonist-independent) activity inherent in the receptor. Here we report that transgenic mice expressing a mutated constitutively active form of the receptor (CAM) show no such phenotype, owing to its modest expression (3-fold above endogenous cardiac beta-adrenergic receptor levels). Surprisingly, treatment of the animals with a variety of beta-adrenergic receptor ligands leads to a 50-fold increase in CAM beta2-adrenergic receptor expression, by stabilizing the CAM beta2-adrenergic receptor protein. Receptor up-regulation leads in turn to marked increases in adenylate cyclase activity, atrial tension determined in vitro, and indices of cardiac contractility determined in vivo. These results illustrate a novel mechanism for regulating physiological responses, i.e., ligand-induced stabilization of a constitutively active but inherently unstable protein.

Sexual relationships, risk behaviour, and condom use in the spread of sexually transmitted infections to heterosexual men.

OBJECTIVE: To examine the effect of patient defined non-regular sexual relationships and other risk behaviours on the incidence of sexually transmitted infections in heterosexual men and the role of condom use in the prevention of their spread. DESIGN: A prospective cross sectional study of sexual behaviour reported by a standardised self administered questionnaire in new patients who presented for screening and diagnosis. SETTING: A genitourinary medicine clinic in west London. SUBJECTS: 957 consecutive newly attending heterosexual men who completed a sexual behaviour questionnaire in 1993/94. MAIN OUTCOME MEASURES: Variables relating to sociodemographic status, sexual behaviour, condom use, sexually transmitted infections and testing for HIV infection, stratified by the reporting of non-regular partners. RESULTS: We found that the 65% of men who reported non-regular sexual partners were more likely to be white collar class (d = 7.5%, 95% CI = 1.3, 13.7) and to have had sexual intercourse with non-United Kingdom born women (d = 7.8%, 95% CI = 3.5, 12.2). They also reported coitarche before 16 years of age (d = 13.4%, 95% CI = 8.0, 18.8) and many more sexual partners both in the last year (d = 13.1%, 95% CI = 10.2, 16.0) and in their lifetime (d = 27.9%, 95% CI = 21.6, 34.2). They were significantly more likely to practise anal intercourse (d = 8.7%, 95% CI = 3.3, 14.1), to smoke (d = 16.3%, 95% CI = 9.8, 22.6), to drink alcohol (d = 4.9%, 95% CI = 1.2, 8.6), and to have chlamydial infection (d = 5.7%, 95% CI = 2.2, 9.2), of which 30% was subclinical. Increasing condom use with regular partners correlated with decreasing incidence of urethral infection (gonorrhoeal and/or chlamydial infection) (p < 0.03) and candidal balanitis (p < 0.03) and a greater likelihood of no infection being detected (p = 0.0002). Use of condoms with non-regular partners was much more frequent than with regular partners (d = 21.4%, 95% CI = 16.7, 26.1). However, we found evidence of oral transmission of urethral gonorrhoea and chlamydial infection among men who reported always using condoms. HIV infection was found in only two men (0.2%), both of whom reported intercourse with non-United Kingdom born women. CONCLUSIONS: Heterosexual men who reported non-regular sexual relationships compensated for their increased risk lifestyle by using condoms more frequently and showed only an increased incidence of chlamydial infection. More consistent condom use with regular partners was significantly associated with the absence of sexually transmitted infection. These findings suggest that transmission between regular partners has been underestimated.

PIP: To investigate the factors associated with sexually transmitted disease transmission, 957 consecutive heterosexual men attending a genitourinary clinic in London, England, in 1993-94 for the first time were enrolled in a prospective cross-sectional study of sexual behavior. 623 men (65%) reported sexual contact with nonregular partners. The respondents who reported nonregular sexual partners were significantly more likely than those who had sex only with regular partners to be white-collar professionals, to have had intercourse with women born outside the UK, to have had first intercourse before the age of 16 years, and to have had more sexual partners in the preceding year and over their lifetime. They were also significantly more likely to smoke, drink, practice anal intercourse, and to be infected with chlamydia. However, 86% of these men reported condom use with nonregular partners. Despite these differences in sexual behavior, men with nonregular sexual partners were at significantly increased risk only for chlamydial infection. Increasing condom use with regular partners was associated with a decreasing incidence of urethral infection and candidal balanitis. There was evidence of oral transmission of urethral gonorrhea and chlamydial infection among men who reported consistent condom use. HIV infection was found in only 2 men (0.2%), both of whom reported sex with women born outside the UK. Overall, these findings indicate that many men who practice high-risk sexual behaviors (multiple partners) take steps to compensate for this risk through condom use.

Inverse agonism and the regulation of receptor number.

Inverse agonists are ligands that preferentially stabilize inactive conformations of G protein-coupled receptors. In a range of systems, sustained treatment with inverse agonists can produce substantially greater upregulation of receptor levels than antagonists. The use of constitutively active mutant receptors can exaggerate this effect but may also allow agonists and antagonists to mimic the effect by preventing denaturation of the mutant receptor polypeptide. In this review Graeme Milligan and Richard Bond consider the basis for these effects and their therapeutic implications.

Essential role of beta-adrenergic receptor kinase 1 in cardiac development and function.

The beta-adrenergic receptor kinase 1 (beta ARK1) is a member of the G protein-coupled receptor kinase (GRK) family that mediates the agonist-dependent phosphorylation and desensitization of G protein-coupled receptors. We have cloned and disrupted the beta ARK1 gene in mice by homologous recombination. No homozygote beta ARK1-/- embryos survive beyond gestational day 15.5. Prior to gestational day 15.5, beta ARK1-/- embryos display pronounced hypoplasia of the ventricular myocardium essentially identical to the "thin myocardium syndrome" observed upon gene inactivation of several transcription factors (RXR alpha, N-myc, TEF-1, WT-1). Lethality in beta ARK1-/- embryos is likely due to heart failure as they exhibit a > 70% decrease in cardiac ejection fraction determined by direct in utero intravital microscopy. These results along with the virtual absence of endogenous GRK activity in beta ARK1-/- embryos demonstrate that beta ARK1 appears to be the predominant GRK in early embryogenesis and that it plays a fundamental role in cardiac development.