Increased myocardial contractility and enhanced exercise function in transgenic mice overexpressing either adenylyl cyclase 5 or 8.

OBJECTIVE: ss-adrenergic receptors (ssARs) are powerful regulators of cardiac function in vivo, activating heterotrimeric G proteins and the effector molecule adenylyl cyclase (AC). Interestingly, cardiac-specific overexpression of different AC isoforms leads to variable changes in cardiac function. Whether AC overexpression affects intrinsic cardiac contractility in an isoform-specific fashion determining a change in exercise capacity is currently unknown. METHODS: To address this issue, we performed load-independent measurements of cardiac systolic and diastolic function by pressure-volume (PV) loop analysis in intact wild-type mice (WT) and transgenic mice overexpressing the AC isoforms 5 or 8. RESULTS: Here we show that cardiac overexpression of either AC5 or AC8 transgenic mice determined an increase in intrinsic cardiac contractility. Interestingly, AC8 transgenic mice displayed a significantly greater increase in cardiac contractility and improved active phase of relaxation. Despite these differences detected by PV loop analysis, both AC5 and AC8 mice showed a marked increase in exercise capacity on treadmill testing. CONCLUSIONS: Our results demonstrate that load-independent measurements of cardiac function are needed to compare different groups of genetically-modified mouse models and to detect subtle AC isoform-specific changes in cardiac performance.

Cyclic AMP compartmentation due to increased cAMP-phosphodiesterase activity in transgenic mice with a cardiac-directed expression of the human adenylyl cyclase type 8 (AC8).

Hearts from AC8TG mice develop a higher contractility (LVSP) and larger Ca2+ transients than NTG mice, with (surprisingly) no modification in L-type Ca2+ channel current (ICa,L) (1). In this study, we examined the cardiac response of AC8TG mice to beta-adrenergic and muscarinic agonists and IBMX, a cyclic nucleotide phosphodiesterase (PDE) inhibitor. Stimulation of LVSP and ICa,L by isoprenaline (ISO, 100 nM) was twofold smaller in AC8TG vs. NTG mice. In contrast, IBMX (100 microM) produced a twofold higher stimulation of ICa,L in AC8TG vs. NTG mice. IBMX (10 microM) increased LVSP by 40% in both types of mice, but contraction and relaxation were hastened in AC8TG mice only. Carbachol (10 microM) had no effect on basal contractility in NTG hearts but decreased LVSP by 50% in AC8TG mice. PDE assays demonstrated an increase in cAMP-PDE activity in AC8TG hearts, mainly due to an increase in the hydrolytic activity of PDE4 and PDE1 toward cAMP and a decrease in the activity of PDE1 and PDE2 toward cGMP. We conclude that cardiac expression of AC8 is accompanied by a rearrangement of PDE isoforms, leading to a strong compartmentation of the cAMP signal that shields L-type Ca2+ channels and protects the cardiomyocytes from Ca2+ overload.

Augmentation of cardiac contractility with no change in L-type Ca2+ current in transgenic mice with a cardiac-directed expression of the human adenylyl cyclase type 8 (AC8).

The beta-adrenergic cascade is severely impaired in heart failure (HF), in part because of a reduction in the activity of the two dominant cardiac adenylyl cyclase (AC) isoforms, AC5 and AC6. Hence, cardiac-directed AC overexpression is a conceivable therapeutic strategy in HF. In this study, we explored the consequences at the cellular and organ level of a cardiac-directed expression of the human AC8 in the transgenic mouse line AC8TG. Unlike AC5 and AC6, which are inhibited by intracellular Ca2+, AC8 is stimulated by Ca2+-calmodulin. Langendorff perfused hearts from AC8TG mice had a twofold higher left ventricular systolic pressure, a 40% faster heart rate, a 37% faster relaxation, and a 30% higher sensitivity to external Ca2+ than nontransgenic control mice (NTG). Cell shortening measured in isolated ventricular myocytes developed 22% faster and relaxed 43% faster in AC8TG than in NTG mice. Likewise, Ca2+ transients measured in fluo-3 AM-loaded myocytes were 30% higher and relaxed 24% faster in AC8TG compared with NTG mice. In spite of the large increase in Ca2+ transients and contraction, expression of AC8 had no effect on the whole-cell L-type Ca2+ current (ICa, L) amplitude. Moreover, ICa, L was unchanged even when AC8 was activated by raising intracellular Ca2+. Thus, cardiac expression of AC8 leads to an increase in cAMP that activates specifically Ca2+ uptake into the sarcoplasmic reticulum but not Ca2+ influx at the sarcolemma, suggesting a strong compartmentation of the cAMP signal.

Nicotine withdrawal syndrome: behavioural distress and selective up-regulation of the cyclic AMP pathway in the amygdala.

Nicotine addiction is a major public health issue. The use of laboratory animal models is a crucial tool in research aiming at understanding the pathophysiological mechanisms of nicotine dependence and at proposing new therapies. In rodents, cessation of nicotine exposure or administration of the nicotinic antagonist mecamylamine induces a nicotine withdrawal syndrome. Antagonist-precipitated withdrawal from other abused drugs such as opiates or cannabinoids has been associated with region-specific modifications of the activity of the cyclic AMP pathway. Here we show that mecamylamine-precipitated nicotine withdrawal in the rat is characterized by an increase in thigmotaxis (time spent in the periphery of an open field) that may be indicative of behavioural distress and can be associated with a selective up-regulation of adenylyl cyclase activity in the amygdala, a region implicated in the regulation of negative affect in response to aversive stimuli, including withdrawal. Adenylyl cyclase activity that is increased during precipitated nicotine withdrawal is stimulated by calcium/calmodulin, as is also the case for opioid and cannabinoid abstinence. This suggests that directly or indirectly mediated increases in the activity of the cyclic AMP pathway could constitute a possible common molecular mechanism underlying neuroadaptive changes following abstinence from different abused drugs.

Novel, not adenylyl cyclase-coupled cannabinoid binding site in cerebellum of mice.

In this study we report data suggesting the presence of a non-CB1, non-CB2 cannabinoid site in the cerebellum of CB1-/- mice. We have carried out [(35)S]GTPgammaS binding experiments in striata, hippocampi, and cerebella of CB1-/- and CB1(+/+) mice with Delta(9)-THC, WIN55,212-2, HU-210, SR141716A, and SR144528. In CB1-/- mice Delta(9)-THC and HU-210 did not stimulate [(35)S]GTPgammaS binding. However, WIN55,212-2 was able to stimulate [(35)S]GTPgammaS binding in cerebella of CB1-/- mice. The maximal effect of this stimulation was 31% that of wild type animals. This effect was reversible neither by CB1 nor CB2 receptor antagonists. Similar results were obtained with the endogenous cannabinoid, anandamide. However, adenylyl cyclase was not inhibited by WIN55,212-2 or anandamide in the CB1(minus sign/minus sign) animals. In striata and hippocampi of CB1-/- mice no [(35)S]GTPgammaS stimulation curve could be obtained with WIN55,212. Our findings suggest that there is a non-CB1 non-CB2 receptor present in the cerebellum of CB1-/- mice.

Studies on the half life time of rat liver transfer RNA species.

The tRNA fraction, extracted from very high speed supernatant fluid, from livers of rats injected with 3H-orotic acid, attained maximum specific activity after a little over 24 hr and, thereafter, decayed with an apparent half life of 5 days. This behaviour of tRNA was indistinguishable from that of liver rRNA and the acid soluble pool. Chromatography of tRNA, doubly labelled during a period of short synthesis and of prolonged decay, on a BD cellulose column, indicated that individual tRNA species turn over at a constant rate with respect to one another.