The discovery of long acting beta2-adrenoreceptor agonists

The design and profile of a series of saligenin containing long acting beta(2)-adrenoreceptor agonists is described. Evaluation of these analogues using a guinea-pig tissue model demonstrates that analogues within this series have significantly longer durations of action than salmeterol and have the potential for a once daily profile in human.     

Relationship of inotropic vagal effects to stretching of the atrium

Isolated left rabbit atria were tested. Preparations are vagal innervated and driven by a constant frequency. Isovolumetric contractions, volumes of the auricles and intraatrial pressures were recorded. With increasing intraatrial pressure the effectivity of peripheral nerve-ending stimulation decreases. There exists a critical value of atrial area. The vagal effectivity will decrease rapidly, if the atrial surface increases higher than this critical value by stretching the auricles.     

Negative inotropic response to cerium in ventricular papillary muscle is mediated by reactive oxygen species

This study was performed with the objective of assessing the mechanical response of the myocardium to different levels of cerium and delineation of the mechanism underlying the mediation of the functional changes. Rat ventricular papillary muscle was used as the experimental model. Isolated papillary muscles were exposed to different concentrations of CeCl₃ and the force of contraction was measured using a force transducer. Experiments have revealed that the negative inotropic response to CeCl₃ was proportional to its concentration. The inotropic changes were found to be completely reversible at concentrations ≤5μM, and partially reversible at higher concentrations. Neutralization of cerium-induced inotropic changes by the superoxide anion scavenger superoxide dismutase (SOD) at concentrations ≤5 μM indicates that the mechanical changes are mediated by reactive oxygen species. At higher concentrations of Ce³⁺, SOD partially reversed the contractile changes. The beneficial effect of SOD was seen only if the muscles were pretreated with the scavenger prior to the addition of cerium chloride.     

A positive inotropic effect of ATP in the human cardiac atrium

We studied contractile effects in isolated electrically driven (1 Hz) atrial preparations from patients undergoing cardiac bypass surgery. ATP concentration dependently (10, 30, and 100 microM) and rapidly decreased force of contraction (negative inotropic effect, NIE) and thereafter more slowly increased force of contraction. The maximum positive inotropic effect (PIE) at 100 microM ATP amounted to 152% of the predrug value (n = 9) and was stable and could be washed out fast and completely. The PIE did not affect time parameters of contraction (time to peak tension and time of relaxation). Moreover, a similar NIE and PIE were noted with adenosine 5'-O-(2-thiotriphosphate) (100 microM). In contrast 2-methyl-thio-ATP did not exert a NIE but only a PIE. In a second set of experiments, preparations were first incubated for 30 min with purinoreceptor antagonists and, in their continuous presence, 100 microM ATP was applied. However, the PIE and NIE of ATP could neither be blocked with suramin (100 and 500 microM), pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (50 microM), nor reactive blue 2 (30, 100, and 500 microM), which are known blockers for subtypes of P(2) receptors, or 1,3-dipropyl-cyclopentvl-xanthine (1 and 10 microM), a subtype (A(1) adenosine) P(1) receptor blocker. Likewise, the inhibitor of phospholipase C (PLC) activity (U-73122) and the inhibitor of adenylate cyclase activity (SQ-022563) (10 microM each) failed to affect the NIE and the PIE of ATP. We tentatively suggest that the PIE of ATP might be mediated via P(2X4)-like receptors. In summary, we describe a novel biphasic effect of ATP on force contraction in the isolated human atrium. It is conceivable that ATP plays a physiological role in the human heart, for instance, after cardiac injury to sustain contractility.     

Mechanism of the negative inotropic effects of alpha 1-adrenoceptor agonists on mouse myocardium

This study was done to identify the mechanism of the alpha1-adrenoceptor (AR) mediated negative inotropic effects of phenylephrine (PE) on adult mouse myocardium. As reported by others, we also found that the nonselective alpha1AR agonist PE produced a negative inotropic effect on ventricular strips from adult mice that was inhibited by the alpha1AAR antagonist 5-methylurapidil (5MU) but not by the alpha1BAR antagonist chloroethylclonidine (CEC) or the alpha1DAR antagonist BMY 7378. The selective alpha1AAR agonist A61603 also produced a negative inotropic effect, which was antagonized by 5MU. Phorbol 12,13-dibutyrate (activator of all PKC isoforms) mimicked the negative inotropic responses to PE and A61603. The negative inotropic effects of PE were inhibited by bisindolylmaleimide (inhibitor of all PKC isoforms) but not by G? 6976 (inhibitor of Ca2+-dependent PKC). Rottlerin, an inhibitor of Ca2+-independent PKCdelta, antagonized the negative inotropic effects of PE and A61603. PE and A61603 increased the translocation of PKCdelta, which was prevented by rottlerin. These data suggest that the alpha1AR-mediated negative inotropy on adult mouse myocardium is signaled by Ca2+-independent PKCdelta.     

The discovery of adamantyl-derived, inhaled, long acting beta(2)-adrenoreceptor agonists

The design and profile of a series of adamantyl-containing long acting beta(2)-adrenoreceptor agonists are described. An optimal pharmacokinetic profile of low oral bioavailability was combined with a strong pharmacology profile when assessed using a guinea pig trachea tissue model. A focus was then placed on developing a robust synthetic route to ensure rapid delivery of material for clinical trials.     

L-type Ca2+ channel facilitation mediated by phosphorylation of the beta subunit by CaMKII

L-type Ca(2+) channels (LTCCs) are major entry points for Ca(2+) in many cells. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is associated with cardiac LTCC complexes and increases channel open probability (P(O)) to dynamically increase Ca(2+) current (I(Ca)) and augment cellular Ca(2+) signaling by a process called facilitation. However, the critical molecular mechanisms for CaMKII localization to LTCCs and I(Ca) facilitation in cardiomyocytes have not been defined. We show CaMKII binds to the LTCC beta(2a) subunit and preferentially phosphorylates Thr498 in beta(2a). Mutation of Thr498 to Ala (T498A) in beta(2a) prevents CaMKII-mediated increases in the P(O) of recombinant LTCCs. Moreover, expression of beta(2a)(T498A) in adult cardiomyocytes ablates CaMKII-mediated I(Ca) facilitation, demonstrating that phosphorylation of beta(2a) at Thr498 modulates native calcium channels. These findings reveal a molecular mechanism for targeting CaMKII to LTCCs and facilitating I(Ca) that may modulate Ca(2+) entry in diverse cell types coexpressing CaMKII and the beta(2a) subunit.     

Desensitization of beta receptor mediated cyclic AMP response of cultured fibroblasts by partial agonists.

Two novel beta-adrenergic myocardial stimulants of general structure: Formula: (see text), where R is a phenyl or benzyl group were investigated for their ability to stimulate and desensitize the cyclic AMP response of C6 glioma cells. Compound ICI 89, 963 (R:phenyl) which elicited less than 1% of the maximum increase in cAMP produced by isoproterenol, was strikingly effective as a desensitizer of the isoproterenol response. This desensitization was markedly reduced by propranolol. Compound ICI 119,033 (R: benzyl) which was a more effective stimulant of cAMP synthesis than ICI 89,963, was also a more effective desensitizer of the isoproterenol response of C6 cells. The kinetics of the desensitization by ICI 89,963 were comparable with those for isoproterenol reaching a maximum in 2 to 3 hours. The data indicate that beta-adrenergic agonists are more potent as desensitizers of the cyclic AMP response than as stimulants of that response.     

The influence of prostaglandins E2 and F2 alpha on electrotropic and inotropic vagal effects on the isolated rabbit atrium

1. PG E2 (2.10(-8) g/ml) produces a positive inotropic effect and lengthens the duration of the action potential at the level of 50% and 90% of repolarization. 2. PG F2 alpha acts negatively inotropic and shortens the action potential at the level of 75% and 90% of repolarization. 3. PG F2 alpha (2.10(-8) g/ml) increases both the electrotropic and inotropic vagal effects, PG E2 (2.10(-8) g/ml) decreases them. 4. PG E2 improves the electromechanical coupling, PG F2 alpha reduces it.     

Modulation of theta-activity in the septo-hippocampal system by the alpha-2-adrenoreceptor agonist clonidine

The influence of the alpha 2-adrenoreceptors agonist clonidine on the neuronal activity of the medial septal area (MS) and hippocampal EEG was studied in unanaesthetized rabbits. A slight and short-term decrease in the theta-rhythm modulation in the MS neuronal activity and/or EEG was revealed in 30.4% of tests after the bilateral intraventricular injection of a small dose of clonidine (0.5 microgram/5 microliters of water). On the contrary, a high dose of clonidine (5 micrograms/5 microliters) substantially enhanced the theta modulation in 100% of tests. The frequency of the theta bursts in the MS increased, on average, by 1.6 +/- 0.18 Hz (from 5.25 +/- 0.19 to 6.8 +/- 0.17 Hz). The regularity of the theta modulation became almost twice higher: the time constant of damping increased from 0.34 +/- 0.04 to 0.60 +/- 0.08 s. Increase in the neuronal activity in the MS produced by the high dose of clonidine was always accompanied by appearance of continuous stable theta waves in the EEG; the spectral power in the theta range increased, on average, by 480 +/- 98%. This strong effect arose suddenly but was relatively short-lasting (12 +/- 3.5 min) and usually abruptly terminated. It is concluded that the noradrenergic system has a double control over the theta oscillations through the alpha 2-adrenoreceptors agonist. In low concentrations the agonist clonidine acts on the high affinity inhibitory presynaptic autoreceptors reducing the noradrenaline release and suppressing the theta activity. In a high dose clonidine predominantly acts on postsynaptic (low affinity) adrenoreceptors substantially increasing the frequency and regularity of the theta bursts in the activity of septal neurons. Presumably, different types of alpha 2-adrenoreceptors participate in regulation of the theta oscillations in different functional states. It is suggested that the noradrenergic locus coeruleus is a functional synergist of the activating reticular formation participating in the urgent phasic activation of the septohippocampal system during the action of sudden strong stimuli.     

Atrioventricular cushion transformation is mediated by ALK2 in the developing mouse heart

Developmental abnormalities in endocardial cushions frequently contribute to congenital heart malformations including septal and valvular defects. While compelling evidence has been presented to demonstrate that members of the TGF-beta superfamily are capable of inducing endothelial-to-mesenchymal transdifferentiation in the atrioventricular canal, and thus play a key role in formation of endocardial cushions, the detailed signaling mechanisms of this important developmental process, especially in vivo, are still poorly known. Several type I receptors (ALKs) for members of the TGF-beta superfamily are expressed in the myocardium and endocardium of the developing heart, including the atrioventricular canal. However, analysis of their functional role during mammalian development has been significantly complicated by the fact that deletion of the type I receptors in mouse embryos often leads to early embryonal lethality. Here, we used the Cre/loxP system for endothelial-specific deletion of the type I receptor Alk2 in mouse embryos. The endothelial-specific Alk2 mutant mice display defects in atrioventricular septa and valves, which result from a failure of endocardial cells to appropriately transdifferentiate into the mesenchyme in the AV canal. Endocardial cells deficient in Alk2 demonstrate decreased expression of Msx1 and Snail, and reduced phosphorylation of BMP and TGF-beta Smads. Moreover, we show that endocardial cells lacking Alk2 fail to delaminate from AV canal explants. Collectively, these results indicate that the BMP type I receptor ALK2 in endothelial cells plays a critical non-redundant role in early phases of endocardial cushion formation during cardiac morphogenesis.     

Isoproterenol response following transfection of the mouse beta 2-adrenergic receptor gene into Y1 cells.

The beta 2-adrenergic receptor (beta 2AR) gene was isolated from a mouse genomic library, sequenced and shown to share 93% identity with the hamster beta 2AR cDNA at the amino acid level. This mouse beta 2AR genomic clone was transfected into the Y1 mouse adrenal cortex tumor cell line. Northern blot and S1 nuclease analysis showed that the beta 2AR-transfected cells expressed an mRNA of the appropriate size to encode the receptor. Membrane receptor number and affinities for various beta-adrenergic agonists demonstrated that the transfected clone encoded a beta 2AR protein product. Incubation of the transfected Y1 cells, which do not normally possess beta 2AR, with the beta 2AR agonist, isoproterenol, resulted in an increase in the rate of steroid secretion by these cells as well as a rapid change in cell morphology. This response was fully blocked by the beta 2AR antagonist, propranolol. Prolonged incubation of the cells with isoproterenol resulted in agonist insensitivity and an 80% reduction in membrane receptor number.     

Contribution of coupling between human myometrial beta2-adrenoreceptor and the BK(Ca) channel to uterine quiescence

The ₂-adrenergic receptor (₂-AR) and the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel have been shown, separately, to be involved in mediating uterine relaxation. Our recent studies reveal that the levels of both ₂-AR and BK_Ca channel proteins in pregnant human myometrium decrease by 50% after the onset of labor. We present direct evidence in support of a structural and functional association between the ₂-AR and the BK_Ca channel in pregnant human myometrium. Localization of both proteins is predominantly plasmalemmal, with 60% of ₂-AR colocalizing with the BK_Ca channel. Coimmunoprecipitation studies indicate that BK_Ca and ₂-AR are structurally linked by direct protein-protein interactions. Functional correlation was confirmed by experiments of human myometrial contractility in which the BK_Ca channel blocker, paxilline, significantly antagonized the relaxant effect of the ₂-AR agonist ritodrine. These novel findings provide an insight into the coupling between the ₂-AR and BK_Ca channel and may have utility in the application of this signaling cascade for therapeutic potential in the management of preterm labor. ₂-adrenergic receptor; myometrium; potassium channel; preterm labor; uterine contraction     

Acute negative inotropic effects of homocysteine are mediated via the endothelium

Previous studies have shown that chronic hyperhomocysteinemia is associated with an adverse cardiac remodeling and heart failure. This study, which utilized coronary-perfused hearts and superfused papillary muscle, was designed to determine whether homocysteine acts acutely to alter cardiac contractile function. Left ventricular developed pressure was used as a measure of systolic function in the Langendorff-perfused heart, whereas isometric developed tension was used in papillary muscle. All preparations were bathed in physiological buffer and paced electrically. Initial results showed that homocysteine elicits a relatively rapid onset (maximum effect observed within 5 min), concentration-dependent (10-300 microM), and moderate negative inotropic action (maximum decrease in tension was approximately 15% of control values) in Langendorff-perfused hearts but not in papillary muscle. In contrast, effluent from homocysteine-treated hearts decreased contractility in papillary muscle, and all inotropic actions were largely eliminated when brief Triton X-100 treatment was utilized to inactivate the coronary endothelium in the intact heart. The homocysteine-induced decrease in contractile function was not antagonized by N(omega)-nitro-l-arginine, a nitric oxide synthase inhibitor, or the cyclooxygenase inhibitor indomethacin. Thus data suggest that pathophysiological concentrations of homocysteine elicit an acute negative inotropic effect on ventricular myocardium that is mediated by a coronary endothelium-derived agent other than nitric oxide or products of cyclooxygenase. Future studies are required to elucidate the mechanism by which homocysteine acts to elicit the release of the proposed endothelial mediator, the identity of the proposed paracrine agent, and the mechanism of its negative inotropic action.     

Direct positive chronotropic action by angiotensin II in the isolated mouse atrium

We observed the direct positive chronotropic effect of angiotensin II in mouse atria and characterized its pharmacological property. C57BL/6J mice were anesthetized with pentobarbital and hearts were quickly excised. Atrial preparations including right and left atrium were isolated and suspended in the organ bath filled with Krebs-Henseleit solution gassed with 95% O2 and 5% CO2. Angiotensin II at concentrations of 10(-10) to 10(-6) M caused concentration-dependent increase in heart rate, and the maximal response was about 13% of that by isoproterenol. The effect was blocked by the selective AT1-receptor antagonist, losartan at concentrations of 10(-6) M, but not by the selective beta-blocker, nadolol at concentration of 10(-5) M. Furthermore, angiotensin I also caused concentration-dependent increase in heart rate, and the effect was blocked by angiotensin converting enzyme (ACE) inhibitor, captopril at concentrations of 10(-6) M. These results suggested that angiotensin I is converted to angiotensin II via ACE system in mice atria, and regulate heart rate through AT1-receptor stimulation, not by beta-adrenergic receptor.