It was investigated how A1‐adenosine receptor overexpression alters the effects of carbachol on force of contraction and beating rate in isolated murine atria. Moreover, the influence of pertussis ...toxin on the inotropic and chronotropic effects of adenosine and carbachol in A1‐adenosine receptor overexpressing atria was studied.
Adenosine and carbachol alone exerted negative inotropic and chronotropic effects in electrically driven left atrium or spontaneously beating right atrium of wild‐type mice.
These effects were abolished or reversed by pre‐treatment of animals with pertussis toxin which can interfere with signal transduction through G‐proteins.
Adenosine and carbachol exerted positive inotropic but negative chronotropic effects in atrium overexpressing A1‐adenosine receptors from transgenic mice.
The positive inotropic effects of adenosine and carbachol were qualitatively unaltered whereas the negative chronotropic effects were abolished or reversed in atrium overexpressing A1‐adenosine receptors after pre‐treatment by pertussis toxin.
Qualitatively similar effects for adenosine and carbachol were noted in the presence of isoprenaline, β‐adrenoceptor agonist.
It is concluded that overexpression of A1‐adenosine receptors also affects the signal transduction of other heptahelical, G‐protein coupled receptors like the M‐cholinoceptor in the heart. The chronotropic but not the inotropic effects of adenosine and carbachol in transgenic atrium were mediated via pertussis toxin sensitive G‐proteins.
British Journal of Pharmacology (2003) 138, 209–217. doi:10.1038/sj.bjp.0705012
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Despite minimal model characterisation Langendorff perfused murine hearts are increasingly employed in cardiovascular research, and particularly in studies of myocardial ischaemia and reperfusion. ...Reported contractility remains poor and ischaemic recoveries variable. We characterised function in C57/BL6 mouse hearts using a ventricular balloon or apicobasal displacement and assessed responses to 10–30 min global ischaemia. We examined the functional effects of pacing, ventricular balloon design, perfusate filtration, Ca2+ and temperature. Contractility was high in isovolumically functioning mouse hearts (measured as the change in pressure with time (+dP/dt), 6000–7000 mmHg s‐1) and was optimal at a heart rate of ±420 beats min‐1, with the vasculature sub‐maximally dilated, and the cellular energy state high. Post‐ischaemic recovery (after 40 min reperfusion) was related to the ischaemic duration: developed pressure recovered by 82 ± 5%, 73 ± 4%, 68 ± 3%, 57 ± 2% and 41 ± 5% after 10, 15, 20, 25 and 30 min ischaemia, respectively. Ventricular compliance and elastance were both reduced post‐ischaemia. Post‐ischaemic recoveries were lower in the apicobasal model (80 ± 4%, 58 ± 7%, 40 ± 3%, 32 ± 7% and 25 ± 5%) despite greater reflow and lower metabolic rate (pre‐ischaemic myocardial O2 consumption (VO2,myo) 127 ± 15 vs. 198 ± 17 μl O2 min‐1 g‐1), contracture, enzyme and purine efflux. Electrical pacing slowed recovery in both models, small ventricular balloons (unpressurised volumes < 50–60 μl) artificially depressed ventricular function and recovery from ischaemia, and failure to filter the perfusion fluid to < 0.45 μm depressed pre‐ and post‐ischaemic function. With attention to these various experimental factors, the buffer perfused isovolumically contracting mouse heart is shown to be stable and highly energised, and to possess a high level of contractility. The isovolumic model is more reliable in assessing ischaemic responses than the commonly employed apicobasal model.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Adenosine and the A1‐adenosine receptor agonist R‐PIA, exerted a negative inotropic effect in isolated, electrically driven left atria of wild‐type mice.
In left atria of mice overexpressing the ...A1‐adenosine receptor, adenosine and R‐PIA exerted a positive inotropic effect.
The positive inotropic effect of adenosine and R‐PIA in transgenic atria could be blocked by the A1‐adenosine receptor antagonist DPCPX.
In the presence of isoprenaline, adenosine exerted a negative inotropic effect in wild‐type atria but a positive inotropic effect in atria from A1‐adenosine receptor overexpressing mice.
The rate of beating in right atria was lower in mice overexpressing A1‐adenosine receptors compared with wild‐type.
Adenosine exerted comparable negative chronotropic effects in right atria from both A1‐adenosine receptor overexpressing and wild‐type mice.
A1‐adenosine receptor overexpression in the mouse heart can reverse the inotropic but not the chronotropic effects of adenosine, implying different receptor‐effector coupling mechanisms.
British Journal of Pharmacology (1999) 128, 1623–1629; doi:10.1038/sj.bjp.0702963
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
1 Department of Pediatrics and the Cardiovascular Research
Center, 2 Department of Biomedical Engineering, University of
Virginia Health Sciences Center, Charlottesville, Virginia 22908; and
3 ... Department of Pharmacology, Merck Research Laboratories, West
Point, Pennsylvania 19486
A 3 adenosine receptors (A 3 ARs) have been
implicated in regulating mast cell function and in cardioprotection
during ischemia-reperfusion injury. The physiological role of
A 3 ARs is unclear due to the lack of widely available
selective antagonists. Therefore, we examined mice with targeted gene
deletion of the A 3 AR together with pharmacological studies
to determine the role of A 3 ARs in myocardial
ischemia-reperfusion injury. We evaluated the functional response to 15-min global ischemia and 30-min reperfusion in
isovolumic Langendorff hearts from A 3 AR /
and wild-type (A 3 AR +/+ ) mice. Loss of
contractile function during ischemia was unchanged, but
recovery of developed pressure in hearts after reperfusion was improved
in A 3 AR / compared with wild-type hearts
(80 ± 3 vs. 51 ± 3% at 30 min). Tissue viability assessed
by efflux of lactate dehydrogenase was also improved in
A 3 AR / hearts (4.5 ± 1 vs. 7.5 ± 1 U/g). The adenosine receptor antagonist BW-A1433 (50 µM) decreased
functional recovery following ischemia in
A 3 AR / but not in wild-type hearts. We also
examined myocardial infarct size using an intact model with 30-min left
anterior descending coronary artery occlusion and 24-h reperfusion.
Infarct size was reduced by over 60% in
A 3 AR / hearts. In summary, targeted deletion
of the A 3 AR improved functional recovery and tissue
viability during reperfusion following ischemia. These data
suggest that activation of A 3 ARs contributes to myocardial injury in this setting in the rodent. Since A 3 ARs are
thought to be present on resident mast cells in the rodent myocardium, we speculate that A 3 ARs may have proinflammatory actions
that mediate the deleterious effects of A 3 AR activation
during ischemia-reperfusion injury.
knockout; myocardial infarction; inflammation; cardiac
protection
SUMMARY
1. Chronotropic and vasodilatory effects of adenosine receptor activation with 2‐chloroadenosine (2‐ClAdo) and β‐adrenoceptor activation with isoproterenol were studied in wild‐type murine ...hearts and transgenic hearts overexpressing the A1 adenosine receptor.
2. Treatment of wild‐type hearts with 2‐ClAdo induced bradycardia (pEC50 6.4±0.2) and vasodilatation (pEC50 7.9±0.1; minimal resistance 2.2±0.2 mmHg/mL per min per g). The A1 receptor‐mediated bradycardia was 20‐fold more sensitive in transgenic hearts (pEC50 7.7±0.2), whereas coronary vasoactivity of 2‐ClAdo was unaltered (pEC50 7.6±0.1).
3. β‐Adrenoceptor stimulation with isoproterenol increased heart rate (pEC50 8.5±0.2; maximal rate 594±23 b.p.m.) and produced vasodilation (pEC50 8.7±0.1; minimal resistance 1.7±0.2 mmHg/mL per min per g) in wild‐type hearts. Treatment with 10 IU/mL adenosine deaminase increased the magnitude of the tachycardia (maximal rate 653±27 b.p.m.) without altering potency (pEC50 8.5±0.1). Antagonism of A1 receptors with 10 nmol/L 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) produced a comparable increase in the magnitude of the chronotropic response (maximal rate 695±26 b.p.m.) without altering potency (pEC50 8.3±0.1).
4. Isoproterenol‐mediated vasodilatation was unaltered by transgenic A1 receptor overexpression. Overexpression of A1 receptors significantly reduced the maximal heart rate during β‐adrenoceptor stimulation by 35% (to 381±28 b.p.m.) without altering potency (pEC50 8.4±0.2). At 10 nmol/L, DPCPX increased the magnitude of the chronotropic response to isoproterenol in transgenic hearts (maximal heart rate 484±36 b.p.m.) without altering potency (pEC50 8.3±0.2).
5. The data show that transgenic A1 receptor overexpression selectively sensitizes the cardiovascular A1 receptor response and that A1 receptor activation by endogenous adenosine depresses the magnitude, but not potency, of the β‐adrenoceptor‐mediated chronotropic response in mouse heart. The A1 receptor‐mediated depression of β‐adrenoceptor responsiveness is non‐competitive (reduced response magnitude with no change in sensitivity). This indicates that A1 receptor activation non‐competitively inhibits effector mechanisms activated by β‐adrenoceptors (e.g. adenylate cyclase) and/or A1 receptors activate unrelated but opposing mechanisms. This inhibitory response may have physiological importance during periods of sympathetic stimulation of cardiac work.
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BFBNIB, DOBA, FSPLJ, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
1 It was investigated how A(1)-adenosine receptor overexpression alters the effects of carbachol on force of contraction and beating rate in isolated murine atria. Moreover, the influence of ...pertussis toxin on the inotropic and chronotropic effects of adenosine and carbachol in A(1)-adenosine receptor overexpressing atria was studied. 2 Adenosine and carbachol alone exerted negative inotropic and chronotropic effects in electrically driven left atrium or spontaneously beating right atrium of wild-type mice. 3 These effects were abolished or reversed by pre-treatment of animals with pertussis toxin which can interfere with signal transduction through G-proteins. 4 Adenosine and carbachol exerted positive inotropic but negative chronotropic effects in atrium overexpressing A(1)-adenosine receptors from transgenic mice. 5 The positive inotropic effects of adenosine and carbachol were qualitatively unaltered whereas the negative chronotropic effects were abolished or reversed in atrium overexpressing A(1)-adenosine receptors after pre-treatment by pertussis toxin. 6 Qualitatively similar effects for adenosine and carbachol were noted in the presence of isoprenaline, beta-adrenoceptor agonist. 7 It is concluded that overexpression of A(1)-adenosine receptors also affects the signal transduction of other heptahelical, G-protein coupled receptors like the M-cholinoceptor in the heart. The chronotropic but not the inotropic effects of adenosine and carbachol in transgenic atrium were mediated via pertussis toxin sensitive G-proteins.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
To identify potential molecular genetic determinants of cardiovascular ischemic tolerance in wild-type and transgenic hearts overexpressing A(1) adenosine receptors (A(1)ARs).
cDNA microarrays were ...used to explore expression of 1824 genes in wild-type hearts and ischemia-tolerant mouse hearts overexpressing A(1)ARs.
Overexpression of A(1)ARs reduced post-ischemic contractile dysfunction, limited arrhythmogenesis, and reduced necrosis by approximately 80% in hearts subjected to 30 min global ischemia 60 min reperfusion. Cardioprotection was abrogated by acute A(1)AR antagonism, and only a small number (19) of genes were modified by A(1)AR overexpression in normoxic hearts. Ischemia-reperfusion significantly altered expression of 75 genes in wild-type hearts (14 induced, 61 down-regulated), including genes for metabolic enzymes, structural/motility proteins, cell signaling proteins, defense/growth proteins, and regulators of transcription and translation. A(1)AR overexpression reversed the majority of gene down-regulation whereas gene induction was generally unaltered. Additionally, genes involved in cell defence, signaling and gene expression were selectively modified by ischemia in transgenic hearts (33 induced, 10 down-regulated), possibly contributing to the protected phenotype. Real-time PCR verified changes in nine selected genes, revealing concordance with array data. Transcription of the A(1)AR gene was also modestly reduced post-ischemia, consistent with impaired functional sensitivity to A(1)AR stimulation
Data are presented regarding the early post-ischemic gene profile of intact heart. Reduced A(1)AR transcription is observed which may contribute to poor outcome from ischemia. A(1)AR overexpression selectively modifies post-ischemic gene expression, potentially contributing to ischemic-tolerance.
Adenosine and the A
1
‐adenosine receptor agonist R‐PIA, exerted a negative inotropic effect in isolated, electrically driven left atria of wild‐type mice.
In left atria of mice overexpressing the A
...1
‐adenosine receptor, adenosine and R‐PIA exerted a positive inotropic effect.
The positive inotropic effect of adenosine and R‐PIA in transgenic atria could be blocked by the A
1
‐adenosine receptor antagonist DPCPX.
In the presence of isoprenaline, adenosine exerted a negative inotropic effect in wild‐type atria but a positive inotropic effect in atria from A
1
‐adenosine receptor overexpressing mice.
The rate of beating in right atria was lower in mice overexpressing A
1
‐adenosine receptors compared with wild‐type.
Adenosine exerted comparable negative chronotropic effects in right atria from both A
1
‐adenosine receptor overexpressing and wild‐type mice.
A
1
‐adenosine receptor overexpression in the mouse heart can reverse the inotropic but not the chronotropic effects of adenosine, implying different receptor‐effector coupling mechanisms.
British Journal of Pharmacology
(1999)
128
, 1623–1629; doi:
10.1038/sj.bjp.0702963
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
We examined myocardial 5′-adenosine monophosphate (5′-AMP) catabolism, adenosine salvage and adenosine responses in perfused guinea pig, rat and mouse heart. M
V
o
2 increased from 71±8 μl O
2/min ...per g in guinea pig to 138±17 and 221±15 μl O
2/min per g in rat and mouse.
V
o
2/beat was 0.42±0.03, 0.50±0.03 and 0.55±0.04 μl O
2/g in guinea pig, rat and mouse, respectively. Resting and peak coronary flows were highest in mouse vs. rat and guinea pig, and peak ventricular pressures and Ca
2+ sensitivity declined as heart mass increased. Net myocardial 5′-AMP dephosphorylation increased significantly as mass declined (3.8±0.5, 9.0±1.4 and 11.0±1.6 nmol/min per g in guinea pig, rat and mouse, respectively). Despite increased 5′-AMP catabolism, coronary venous adenosine was similar in guinea pig, rat and mouse (45±8, 69±10 and 57±14 nM, respectively). Comparable venous adenosine was achieved by increased salvage vs. deamination: 64%, 41% and 39% of adenosine formed was rephosphorylated while 23%, 46%, and 50% was deaminated in mouse, rat and guinea pig, respectively. Moreover, only 35–45% of inosine and its catabolites derive from 5′-AMP (vs. IMP) dephosphorylation in all species. Although post-ischemic purine loss was low in mouse (due to these adaptations), functional tolerance to ischemia decreased with heart mass. Cardiovascular sensitivity to adenosine also differed between species, with A
1 receptor sensitivity being greatest in mouse while A
2 sensitivity was greatest in guinea pig. In summary: (i) cardiac 5′-AMP dephosphorylation,
V
o
2, contractility and Ca
2+ sensitivity all increase as heart mass falls; (ii) adaptations in adenosine salvage vs. deamination limit purine loss and yield similar adenosine levels across species; (iii) ischemic tolerance declines with heart mass; and (iv) cardiovascular sensitivity to adenosine varies, with increasing A
2 sensitivity relative to A
1 sensitivity in larger hearts.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, PNG, SAZU, SBCE, SBJE, UL, UM, UPUK
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