John Patrick Headrick

Estrogen improves biochemical and neurologic outcome following traumatic brain injury in male rats, but not in females

Phosphorus magnetic resonance spectroscopy was used in conjunction with neurologic motor function tests to assess the effects of estrogen on biochemical and neurologic outcome following traumatic brain injury in male and female rats. Male (n = 18) and female (n = 18) rats were randomly assigned into three groups, and 4 h prior to injury received either 17 beta-estradiol (144 micrograms/kg intraperitoneally), equal volume vehicle (30% ethanol in saline), or no treatment. Traumatic brain injury was induced at 2.8 atm using a fluid percussion injury device, and animals monitored for 4 h using phosphorus magnetic resonance spectroscopy to determine brain intracellular pH, free magnesium concentration and cytosolic phosphorylation potential. Males treated with estrogen demonstrated a significant improvement in free magnesium concentration, and slightly improved values of cytosolic phosphorylation potential after trauma when compared to controls. There was also a significant improvement in post-traumatic motor function at 1 week after trauma. In contrast, estrogen treatment in females lowered cytosolic phosphorylation potential after trauma, but did not affect free magnesium concentration after trauma. Mortality in all female groups was significantly worse than in males. We conclude that estrogen is protective in males, but exacerbates brain injury in females through effects mediated by estrogen receptor binding.

Cardiovascular adenosine receptors: expression, actions and interactions.

Intra- and extracellular adenosine levels rise in response to physiological stimuli and with metabolic/energetic perturbations, inflammatory challenge and tissue injury. Extracellular adenosine engages members of the G-protein coupled adenosine receptor (AR) family to mediate generally beneficial acute and adaptive responses within all constituent cells of the heart. In this way the four AR sub-types-A1, A2A, A2B, and A3Rs-regulate myocardial contraction, heart rate and conduction, adrenergic control, coronary vascular tone, cardiac and vascular growth, inflammatory-vascular cell interactions, and cellular stress-resistance, injury and death. The AR sub-types exert both distinct and overlapping effects, and may interact in mediating these cardiovascular responses. The roles of the ARs in beneficial modulation of cardiac and vascular function, growth and stress-resistance render them attractive therapeutic targets. However, interactions between ARs and with other receptors, and their ubiquitous distribution throughout the body, can pose a challenge to the implementation of site- and target-specific AR based pharmacotherapy. This review outlines cardiovascular control by adenosine and the AR family in health and disease, including interactions between AR sub-types within the heart and vessels.

Loss of Caveolin-1 Accelerates Neurodegeneration and Aging

Background The aged brain exhibits a loss in gray matter and a decrease in spines and synaptic densities that may represent a sequela for neurodegenerative diseases such as Alzheimers. Membrane/lipid rafts (MLR), discrete regions of the plasmalemma enriched in cholesterol, glycosphingolipids, and sphingomyelin, are essential for the development and stabilization of synapses. Caveolin-1 (Cav-1), a cholesterol binding protein organizes synaptic signaling components within MLR. It is unknown whether loss of synapses is dependent on an age-related loss of Cav-1 expression and whether this has implications for neurodegenerative diseases such as Alzheimers disease. Methodology/Principal Findings We analyzed brains from young (Yg, 3-6 months), middle age (Md, 12 months), aged (Ag, >18 months), and young Cav-1 KO mice and show that localization of PSD-95, NR2A, NR2B, TrkBR, AMPAR, and Cav-1 to MLR is decreased in aged hippocampi. Young Cav-1 KO mice showed signs of premature neuronal aging and degeneration. Hippocampi synaptosomes from Cav-1 KO mice showed reduced PSD-95, NR2A, NR2B, and Cav-1, an inability to be protected against cerebral ischemia-reperfusion injury compared to young WT mice, increased Aβ, P-Tau, and astrogliosis, decreased cerebrovascular volume compared to young WT mice. As with aged hippocampi, Cav-1 KO brains showed significantly reduced synapses. Neuron-targeted re-expression of Cav-1 in Cav-1 KO neurons in vitro decreased Aβ expression. Conclusions Therefore, Cav-1 represents a novel control point for healthy neuronal aging and loss of Cav-1 represents a non-mutational model for Alzheimers disease.

Ischaemic tolerance in aged mouse myocardium: the role of adenosine and effects of A1 adenosine receptor overexpression

The genesis of the ischaemia intolerant phenotype in aged myocardium is poorly understood. We tested the hypothesis that impaired adenosine‐mediated protection contributes to ischaemic intolerance, and examined whether this is countered by A1 adenosine receptor (A1AR) overexpression. Responses to 20 min ischaemia and 45 min reperfusion were assessed in perfused hearts from young (2–4 months) and moderately aged (16–18 months) mice. Post‐ischaemic contractility was impaired by ageing with elevated ventricular diastolic (32 ± 2 vs. 18 ± 2 mmHg in young) and reduced developed (37 ± 3 vs. 83 ± 6 mmHg in young) pressures. Lactate dehydrogenase (LDH) loss was exaggerated (27 ± 2 vs. 16 ± 2 IU g−1in young) whereas the incidence of tachyarrhythmias was similar in young (15 ± 1 %) and aged hearts (16 ± 1 %). Functional analysis confirmed equipotent effects of 50 μm adenosine at A1 and A2 receptors in young and aged hearts. Nonetheless, while 50 μm adenosine improved diastolic (5 ± 1 mmHg) and developed pressures (134 ± 7 mmHg) and LDH loss (6 ± 2 IU g−1) in young hearts, it did not alter these variables in the aged group. Adenosine did attenuate arrhythmogenesis for both ages (to ∼10 %). In contrast to adenosine, 50 μm diazoxide reduced ischaemic damage and arrhythmogenesis for both ages. Contractile and anti‐necrotic effects of adenosine were limited by 100 μm 5‐hydroxydecanoate (5‐HD) and 3 μm chelerythrine. Anti‐arrhythmic effects were limited by 5‐HD but not chelerythrine. Non‐selective (100 μm 8‐sulfophenyltheophylline) and A1‐selective (150 nm 8‐cyclopentyl‐1,3‐dipropylxanthine) adenosine receptor antagonism impaired ischaemic tolerance in young but not aged hearts. Quantitative real‐time PCR and radioligand analysis indicated that impaired protection is unrelated to changes in A1AR mRNA transcription, or receptor density (∼8 fmol mg−1 protein in both age groups). However, A1AR overexpression improved tolerance for both ages, restoring adenosine‐mediated protection. These data reveal impaired protection via exogenous and endogenous adenosine contributes to ischaemic intolerance with ageing. This is independent of A1AR expression, and involves ineffective activation of a 5‐HD‐/diazoxide‐sensitive process. The effects of A1AR overexpression indicate that the age‐related failure in signalling can be overcome.

Adenosine and its receptors in the heart: Regulation, retaliation and adaptation

The purine nucleoside adenosine is an important regulator within the cardiovascular system, and throughout the body. Released in response to perturbations in energy state, among other stimuli, local adenosine interacts with 4 adenosine receptor sub-types on constituent cardiac and vascular cells: A(1), A(2A), A(2B), and A(3)ARs. These G-protein coupled receptors mediate varied responses, from modulation of coronary flow, heart rate and contraction, to cardioprotection, inflammatory regulation, and control of cell growth and tissue remodeling. Research also unveils an increasingly complex interplay between members of the adenosine receptor family, and with other receptor groups. Given generally favorable effects of adenosine receptor activity (e.g. improving the balance between myocardial energy utilization and supply, limiting injury and adverse remodeling, suppressing inflammation), the adenosine receptor system is an attractive target for therapeutic manipulation. Cardiovascular adenosine receptor-based therapies are already in place, and trials of new treatments underway. Although the complex interplay between adenosine receptors and other receptors, and their wide distribution and functions, pose challenges to implementation of site/target specific cardiovascular therapy, the potential of adenosinergic pharmacotherapy can be more fully realized with greater understanding of the roles of adenosine receptors under physiological and pathological conditions. This review addresses some of the major known and proposed actions of adenosine and adenosine receptors in the heart and vessels, focusing on the ability of the adenosine receptor system to regulate cell function, retaliate against injurious stressors, and mediate longer-term adaptive responses.

Clinical cardioprotection and the value of conditioning responses

Adjunctive cardioprotective strategies for ameliorating the reversible and irreversible injuries with ischemia-reperfusion (I/R) are highly desirable. However, after decades of research, the promise of clinical cardioprotection from I/R injury remains poorly realized. This may arise from the challenges of trialing and effectively translating experimental findings from laboratory models to patients. One can additionally consider whether features of the more heavily focused upon candidates could limit or preclude therapeutic utility and thus whether we might shift attention to alternate strategies. The phenomena of preconditioning and postconditioning have proven fertile in identification of experimental means of cardioprotection and are the most intensely interrogated responses in the field. However, there is evidence these processes, which share common molecular signaling elements and end effectors, may be poor choices for clinical exploitation. This includes evidence of age dependence, limiting efficacy in target aged or senescent hearts; refractoriness to conditioning stimuli in diseased myocardium; interference from a variety of relevant pharmaceuticals; inadvertent induction of these responses by prior ischemia or commonly used drugs, precluding further benefit; and sex dependence of protective signaling. This review focuses on these features, raising questions about current research strategies, and the suitability of these widely studied phenomena as rational candidates for clinical translation.

Adenosine receptor subtypes mediating coronary vasodilation in rat hearts.

Adenosine receptor-mediated coronary vasodilation was studied in isolated hearts from young (1–2 months) and mature (12–18 months) Wistar rats. The nonselective agonist 5´-N-ethylcarboxamidoadenosine (NECA) induced biphasic concentration-dependant dilation with similar potencies in both age groups (p < 0.05). Despite similar potencies, responses to NECA were significantly depressed by 50% with age. NECA-mediated dilation was unaltered by selective A1 adenosine receptor (A1AR) antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 100 n M) or A2A adenosine receptor (A2AAR) antagonist 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH 58261, 100 n M). However, the A2B adenosine receptor (A2B AR) selective antagonist alloxazine (10 &mgr;M) significantly reduced response magnitude to NECA in both age groups. Concentration–response curves to N6-2-(4-aminophenyl) ethyladenosine (APNEA) induced biphasic concentration-dependent dilation in hearts from young animals. In the presence of the three combined antagonists, 1 &mgr;M DPCPX, 100 n M SCH 58261, and 1 &mgr;M alloxazine, the response magnitude was significantly attenuated (p < 0.05). The addition of the A3 adenosine receptor (A3AR) antagonist 3-ethyl-5-benzyl-2-methyl-4-phenylethyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS1191, 100 n M) to the combined antagonists further attenuated vasodilator responses to APNEA. The results suggest that multiple adenosine receptor subtypes mediate dilation in the rat coronary circulation. NECA mediates vasodilation via the A2BAR subtype, while dilator responses to APNEA in the presence and absence of A1, A2, and A3 AR antagonists provide evidence for a vasodilator role for A3 ARs in rat coronary circulation. The magnitude of the coronary dilator response is reduced with age and does not involve A2A or A1 ARs.

Functional properties and responses to ischaemia‐reperfusion in Langendorff perfused mouse heart

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.

Adenosine-mediated cardioprotection in ischemic-reperfused mouse heart.

We investigated the roles of A 1 , A 2A , or A 3 receptors and purine salvage in cardioprotection with exogenous adenosine, and tested whether A 2A -mediated reductions in perfusion pressure modify post-ischemic recovery. Treatment with 10 −5 or 5 × 10 −5 M adenosine improved contractile recovery from 20 min ischemia 45 min reperfusion in isolated mouse hearts. Protection was attenuated by adenosine kinase inhibition (10 −5 M iodotubercidin) and receptor antagonism (5 × 10 −5 M 8-&rgr;-sulfophenyltheophylline, 8-SPT). Enzyme efflux mirrored contractile recoveries. A 3 agonism with 10 −7 M 2-chloro-N 6 -(3-iodobenzyl)-adenosine-5´-N-methyluronamide (Cl-IB-MECA) improved ischemic tolerance whereas A 1 agonism with 5 × 10 −8 M N 6 -cyclopentyladenosine (CPA) and A 2A agonism with 10 −9 M 2-[p-(2-carboxyethyl) phenethylamino]-5´-N-ethylcarboxamidoadenosine (CGS21680) or 2 × 10 −8 M methyl-4-(3-{9-[4S,5S,2R,3R)-5-(N-ethylcarbamoyl)-3,4-dihydroxyoxolan-2-yl]-6-aminopurin-2-yl)}prop-2-ynyl) cyclohexane-carboxylate (ATL-146e) were ineffective. Protection via A 1 receptor overexpression was enhanced by adenosine, but unaltered by A 1 or A 2A agonists. Finally, post-ischemic dysfunction in hearts perfused at constant flow was dependent on coronary pressure, with A 2A AR-mediated reductions in pressure reducing diastolic contracture, and elevated perfusion pressure worsening contracture. Data indicate that cardioprotection with exogenous adenosine in asanguinous hearts involves purine salvage and activation of A 3 but not A 1 or A 2A receptors.

Genetic Deletion of the A1 Adenosine Receptor Limits Myocardial Ischemic Tolerance

Adenosine receptors may be important determinants of intrinsic ischemic tolerance. Genetically modified mice were used to examine effects of global A1 adenosine receptor (A1AR) knockout (KO) on function and ischemic tolerance in perfused mouse hearts. Baseline contractile function and heart rate were unaltered by A1AR KO, which was shown to abolish the negative chronotropic effects of 2-chloroadenosine (A1AR-mediated) without altering A2 adenosine receptor–mediated coronary dilation. Tolerance to 25 minutes global normothermic ischemia (followed by 45 minutes reperfusion) was significantly limited by A1AR KO, with impaired contractile recovery (reduced by ≈25%) and enhanced lactate dehydrogenase (LDH) efflux (increased by ≈100%). Functional effects of A1AR KO involved worsened systolic pressure development with little to no change in diastolic dysfunction. In contrast, cardiac specific A1AR overexpression enhanced ischemic tolerance with a primary action on diastolic dysfunction. Nonselective receptor agonism (10 &mgr;mol/L 2-chloroadenosine) protected wild-type and also A1AR KO hearts (albeit to a lesser extent), implicating protection via subtypes additional to A1ARs. However, A1AR KO abrogated effects of 2-chloroadenosine on ischemic contracture and diastolic dysfunction. These data are the first demonstrating global deletion of the A1AR limits intrinsic myocardial resistance to ischemia. Data indicate the function of intrinsically activated A1ARs appears primarily to be enhancement of postischemic contractility and limitation of cell death.