Cardiac function in mice overexpressing the beta-adrenergic receptor kinase or a beta ARK inhibitor.

Transgenic mice were created with cardiac-specific overexpression of the beta-adrenergic receptor kinase-1 (beta ARK1) or a beta ARK inhibitor. Animals overexpressing beta ARK1 demonstrated attenuation of isoproterenol-stimulated left ventricular contractility in vivo, dampening of myocardial adenylyl cyclase activity, and reduced functional coupling of beta-adrenergic receptors. Conversely, mice expressing the beta ARK inhibitor displayed enhanced cardiac contractility in vivo with or without isoproterenol. These animals demonstrate the important role of beta ARK in modulating in vivo myocardial function. Because increased amounts of beta ARK1 and diminished cardiac beta-adrenergic responsiveness characterize heart failure, these animals may provide experimental models to study the role of beta ARK in heart disease.

Enhanced myocardial function in transgenic mice overexpressing the beta 2-adrenergic receptor.

Transgenic mice were created with cardiac-specific overexpression of the beta 2-adrenergic receptor. This resulted in increased basal myocardial adenylyl cyclase activity, enhanced atrial contractility, and increased left ventricular function in vivo; these parameters at baseline in the transgenic animals were equal to those observed in control animals maximally stimulated with isoproterenol. These results illustrate a useful approach for studying the effect of gene expression on cardiac contractility. Because chronic heart failure in humans is accompanied by a reduction in the number of myocardial beta-adrenergic receptors and in inotropic responsiveness, these results suggest a potential gene therapy approach to this disease state.

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.

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.

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.

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.

Agonist and antagonist characterization of a putative adrenoceptor with distinct pharmacological properties from the alpha- and beta-subtypes.

1. Experiments were done to characterize a putative adrenoceptor which functions to inhibit longitudinal muscle tension development in the guinea-pig ileum. Several phenylethylamine based agonists were investigated: BRL 37344, (-)-isoprenaline, (+)-isoprenaline, noradrenaline, adrenaline, and fenoterol. Propranolol and nadolol were tested as antagonists. Agonist-induced inhibition of the contractile response to histamine was measured under equilibrium conditions with alpha-adrenoceptors and muscarinic cholinoceptors inhibited. 2. Inhibitory responses were obtained to (-)-isoprenaline and BRL 37344 that were resistant to beta-adrenoceptor blockage with propranolol (5 microM) and nadolol (10 microM). These resistant responses were antagonized by much higher concentrations of nadolol (30 to 1000 microM) yielding apparent pA2 values for nadolol of 4.31 with (-)-isoprenaline as the agonist, and 4.68 with BRL 37344 as the agonist. Similar apparent pA2 values for nadolol at the putative adrenoceptor were obtained with noradrenaline (4.79), adrenaline (4.68), and fenoterol (4.38). 3. The order and relative potency of agonists at the putative adrenoceptor was: BRL 37344 (20) greater than (-)-isoprenaline (8) greater than noradrenaline (1) greater than adrenaline (0.5) greater than fenoterol (0.35) greater than (+)-isoprenaline (0.27). 4. The resistance to blockade by propranolol (5 microM), the low affinity of nadolol, and the order and relative potency of agonists, suggest the presence of an adrenoceptor with distinct pharmacological characteristics from currently defined alpha- and beta-adrenoceptors.

A response to isoprenaline unrelated to alpha- and beta-adrenoceptor agonism.

The hypothesis that beta-adrenoceptor agonism might explain a reported lack of competitive antagonism between alpha 2-adrenoceptor antagonists and agonists of the phenylethylamine class was tested in the electrically field stimulated ileum of the guinea-pig. The beta-adrenoceptor agonist, isoprenaline, was used as the phenylethylamine and inhibition of 'twitch' response evoked by cholinergic stimulation was measured. In the presence of idazoxan (3 microM), to block inhibitory alpha 2-adrenoceptors, propranolol (0.1 to 5.0 microM) failed to act competitively toward isoprenaline. Isoprenaline responses totally resistant to inhibition by propranolol were obtained. As inhibitory alpha 1-adrenoceptors are absent from guinea-pig ileum, a recognition site distinct from the currently defined alpha- and beta-adrenoceptors is postulated. Agonism by phenylethylamine based agonists at this site may explain their inability to act competitively with alpha- and beta-adrenoceptor antagonists.

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.

Marked enhancement in myocardial function resulting from overexpression of a human beta-adrenergic receptor gene.

Transgenic mice with intense cardiac expression of a human beta-adrenergic receptor gene were engineered and shown to display marked improvements in baseline myocardial and left ventricular function. Heart/body weight ratios and histologic appearance were not found to be significantly altered, suggesting that receptor gene expression did not induce pathologic changes. Given the substantial reduction in beta-adrenergic receptor density and resultant reduction in inotropic responsiveness observed in chronic heart failure, these findings represent a novel approach for increasing myocardial function with important clinical implications.

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.

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.

Evidence for a receptor mediated action of norepinephrine distinct from alpha- and beta-adrenoceptors.

The mode of action of (-) norepinephrine (NE) and UK-14,304-18 has been investigated using the cholinergically-evoked 'twitch' response of the electrically stimulated guinea-pig ileum. St 587 and benextramine were employed as antagonists. St 587 acted as a competitive antagonist toward UK-14,304-18, yielding an apparent pA2 value of 7.3. In contrast, St 587 failed to act competitively toward NE. Similarly, benextramine (1 X 10(-5) mol/l) blocked the inhibitory responses to UK-14,304-18 but was considerably less active toward NE. Remaining responses to NE after benextramine were not antagonized by St 587, even at a concentration of 3 X 10(-5) mol/l. It is postulated that NE acts to inhibit the 'twitch' response be evoking two different receptor-mediated events: 1. agonism at the alpha 2-adrenoceptor and 2. agonism at a site which is distinct from the alpha- and beta-subtypes. In the concentrations studied, UK-14,304-18, St 587 and benextramine are postulated to lack affinity for the proposed site. The effect of NE and UK-14,304-18 was also investigated on the contractile responses to exogenously applied histamine. These experiments were done in the presence of muscarinic cholinergic and adrenoceptor blockade. NE inhibited responses to histamine but UK-14,304-18 was inactive. Furthermore, the inhibitory action of NE was stereoselective with the (-) form being 25 times more potent than the (+) enantiomer. These findings suggest the presence of a receptor site for NE which is distinct from cholinergic mechanisms and established alpha and beta-adrenoceptors.

An inhibitory prejunctional 5-HT1-like receptor in the isolated perfused rat kidney. Apparent distinction from the 5-HT1A, 5-HT1B and 5-HT1C subtypes.

The present study has identified a receptor for 5-hydroxytryptamine (5-HT) which functions to inhibit the stimulus-induced release of [3H] noradrenaline following sympathetic periarterial nerve stimulation to the isolated perfused rat kidney. In addition to 5-HT (IC30 = 4.5 X 10(-8) mol/l), both 5-carboxamidotryptamine (IC30 = 8 X 10(-9) mol/l) and 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl) indole (RU-24969, IC30 = 2.5 X 10(-7) mol/l) acted as agonists whereas 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) was inactive. The inhibitory effect of 5-HT on the electrically-evoked release of tritium was antagonized in a concentration-dependent manner by methiothepin (IC50 = 4 X 10(-9) mol/l), metergoline (IC50 = 4 X 10(-8) mol/l) and methysergide (IC50 = 1.3 X 10(-7) mol/l) but not by cyproheptadine, ketanserin, mesulergine, (-)-propranolol, (+/-)-pindolol, (+/-)-cyanopindolol, metoclopramide or phentolamine. It is concluded that the receptor to 5-HT conforms to general criteria defining 5-HT1-like receptors but at the present time the receptor site cannot be fitted to the designated 5-HT1A, 5-HT1B or 5-HT1C subtypes.

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).

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.

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.

Trends in sexual behaviour and risk factors for HIV infection among homosexual men, 1984-7.

To assess whether the spread of infection with HIV can be reduced by changes in behaviour among groups most at risk because of their sexual practices sexual behaviour was monitored among 1050 homosexual men tested for HIV infection at a genitourinary medicine clinic in west London from November 1984 to September 1987. Four cohorts, defined by date of presentation, were studied by questionnaire at their presentation, and blood samples were analysed. Between the first and last cohorts there was a considerable fall in the proportion reporting casual relationships (291/329 (88%) v 107/213 (50%] and high risk activities, such as anoreceptive intercourse with casual partners (262/291 (90%) v 74/106 (70%], with the greatest changes occurring before the government information campaign began in 1986. Nevertheless, half of the men in the last cohort studied reported having casual partners. Multiple logistic regression showed that behavioural risk factors for HIV infection most closely resembled those for hepatitis B and that previous sexually transmitted diseases (syphilis, hepatitis B, and anogenital herpes) were themselves independent risk factors. A history of syphilis ranked above anoreceptive intercourse as the strongest predictor of HIV infection. Actively bisexual men showed a much lower prevalence of HIV infection (3/57, 5%) than exclusively homosexual men (113/375, 30%). Sexual behaviour among homosexual men changed during the period studied, and the incidence of HIV infection fell, although more education programmes directed at homosexual men are needed to re-emphasise the dangers of infection.