Impaired coronary metabolic dilation in the metabolic syndrome is linked to mitochondrial dysfunction and mitochondrial DNA damage.

Mitochondrial dysfunction in obesity and diabetes can be caused by excessive production of free radicals, which can damage mitochondrial DNA. Because mitochondrial DNA plays a key role in the production of ATP necessary for cardiac work, we hypothesized that mitochondrial dysfunction, induced by mitochondrial DNA damage, uncouples coronary blood flow from cardiac work. Myocardial blood flow (contrast echocardiography) was measured in Zucker lean (ZLN) and obese fatty (ZOF) rats during increased cardiac metabolism (product of heart rate and arterial pressure, i.v. norepinephrine). In ZLN increased metabolism augmented coronary blood flow, but in ZOF metabolic hyperemia was attenuated. Mitochondrial respiration was impaired and ROS production was greater in ZOF than ZLN. These were associated with mitochondrial DNA (mtDNA) damage in ZOF. To determine if coronary metabolic dilation, the hyperemic response induced by heightened cardiac metabolism, is linked to mitochondrial function we introduced recombinant proteins (intravenously or intraperitoneally) in ZLN and ZOF to fragment or repair mtDNA, respectively. Repair of mtDNA damage restored mitochondrial function and metabolic dilation, and reduced ROS production in ZOF; whereas induction of mtDNA damage in ZLN reduced mitochondrial function, increased ROS production, and attenuated metabolic dilation. Adequate metabolic dilation was also associated with the extracellular release of ADP, ATP, and H2O2 by cardiac myocytes; whereas myocytes from rats with impaired dilation released only H2O2. In conclusion, our results suggest that mitochondrial function plays a seminal role in connecting myocardial blood flow to metabolism, and integrity of mtDNA is central to this process.

Reduced adenosine release from the aged mammalian heart.

Adenosine (ADO) released in the heart results in enhanced coronary blood flow and reduced catecholamine release and myocardial responsiveness to adrenergic stimulation (anti-adrenergic action). ADO release from the adrenergic-stimulated aged heart is less than that from the young adult heart. Because adrenergic signaling in the aged heart is impaired, this study was conducted to determine if reduced ADO release from the aged heart results from this reduced adrenergic responsiveness. Hearts of 3-4 months (young adult) and 21-22 months (aged) Fischer-344 rats were perfused with ADO deamination and re-phosphorylation inhibited. Coronary effluent ADO levels were determined. Cellular-free ADO levels with and without sodium acetate (NaAc)-induced mitochondrial AMP synthesis were assessed using formed S-adenosylhomocysteine (SAH) in L-homocysteine thiolactone (L-HC)-treated hearts. The activities of SAH-hydrolase were determined. Aged heart ADO release was 61% less than from young hearts. NaAc augmented young heart ADO release by 104%, while that of aged hearts remained unchanged. SAH synthesis was 51% and 56% lower in the aged heart in the absence and presence of NaAc, respectively, despite an 89% greater SAH hydrolase activity found in the aged hearts. Since synthesized AMP may be diverted to IMP and ultimately inosine by AMP deaminase, inosine release was determined. Aged heart inosine levels in the absence and presence of NaAc were 74% and 59% less than for the young hearts. It is concluded that a reduced mitochondrial AMP synthesis is in part responsible for the attenuation in ADO release from the adrenergic-stimulated aged heart.

Crosstalk between adenosine A1 and ß1-adrenergic receptors regulates translocation of PKCε in isolated rat cardiomyocytes.

Adenosine A(1) receptor (A(1)R)-induced translocation of PKCε to transverse (t) tubular membranes in isolated rat cardiomyocytes is associated with a reduction in ß(1)-adrenergic-stimulated contractile function. The PKCε-mediated activation of protein kinase D (PKD) by endothelin-1 is inhibited by ß(1)-adrenergic stimulated protein kinase A (PKA) suggesting a similar mechanism of A(1)R signal transduction modulation by adrenergic agonists may exist in the heart. We have investigated the influence of ß(1)-adrenergic stimulation on PKCε translocation elicited by A(1)R. Immunofluorescence imaging and Western blotting with PKCε and ß-COP antibodies were used to quantify the co-localization of PKCε and t-tubular structures in isolated rat cardiomyocytes. The A(1)R agonist CCPA increased the co-localization of PKCε and t-tubules as detected by imaging. The ß(1)-adrenergic receptor agonist isoproterenol (ISO) inhibited this effect of CCPA. Forskolin, a potent activator of PKA, mimicked, and H89, a pharmacological PKA inhibitor, and PKI, a membrane-permeable PKA peptide PKA inhibitor, attenuated the negative effect of ISO on the A(1)R-mediated PKCε translocation. Western blotting with isolated intact hearts revealed an increase in PKCε/ß-COP co-localization induced by A(1)R. This increase was attenuated by the A(1)R antagonist DPCPX and ISO. The ISO-induced attenuation was reversed by H89. It is concluded that adrenergic stimulation inhibits A(1)R-induced PKCε translocation to the PKCε anchor site RACK2 constituent of a coatomer containing ß-COP and associated with the t-tubular structures of the heart. In that this translocation has been previously associated with the antiadrenergic property of A(1)R, it is apparent that the interactive effects of adenosine and ß(1)-adrenergic agonists on function are complex in the heart.

Myocardial adenosine A(1)-receptor-mediated adenoprotection involves phospholipase C, PKC-epsilon, and p38 MAPK, but not HSP27.

Adenosine via an adenosine A(1) receptor (A(1)R) is a negative feedback inhibitor of adrenergic stimulation in the heart, protecting it from toxic effects of overstimulation. Stimulation of the A(1)R results in the activation of G(i) protein, release of free Gbetagamma-subunits, and activation/translocation of PKC-epsilon to the receptor for activated C kinase 2 protein at the Z-line of the cardiomyocyte sarcomere. Using an anti-Gbetagamma peptide, we investigated the role of these subunits in the A(1)R stimulation of phospholipase C (PLC), with the premise that the resulting diacylglycerol provides for the activation of PKC-epsilon. Inositol 1,4,5-triphosphate release was an index of PLC activity. Chlorocyclopentyl adenosine (CCPA), an A(1)R agonist, increased inositol 1,4,5-triphosphate production by 273% in mouse heart homogenates, an effect absent in A(1)R knockout hearts and inhibited by anti-Gbetagamma peptide. In a second study, p38 MAPK and heat shock protein 27 (HSP27), found by others to be associated with the loss of myocardial contractile function, were postulated to play a role in the actions of A(1)R. Isoproterenol, a beta-adrenergic receptor agonist, increased the Ca(2+) transient and sarcomere shortening magnitudes by 36 and 49%, respectively. In the rat cardiomyocyte, CCPA significantly reduced these increases, an action blocked by the p38 MAPK inhibitor SB-203580. While CCPA significantly increased the phosphorylation of HSP27, this action was inhibited by isoproterenol. These data indicate that the activation of PKC-epsilon by A(1)R results from the activation of PLC via free Gbetagamma-subunits released upon A(1)R-induced dissociation of G(i)alphabetagamma. Attenuation of beta-adrenergic-induced contractile function by A(1)R may involve the activation of p38 MAPK, but not HSP27.

Adenoprotection of the heart involves phospholipase C-induced activation and translocation of PKC-epsilon to RACK2 in adult rat and mouse.

Adenosine protects the heart from adrenergic overstimulation. This adenoprotection includes the direct anti-adrenergic action via adenosine A(1) receptors (A(1)R) on the adrenergic signaling pathway. An indirect A(1)R-induced attenuation of adrenergic responsiveness involves the translocation of PKC-epsilon to t-tubules and Z-line of cardiomyocytes. We investigated with sarcomere imaging, immunocytochemistry imaging, and coimmunoprecipitation (co-IP) whether A(1)R activation of PKC-epsilon induces the kinase translocation to receptor for activated C kinase 2 (RACK2) in isolated rat and mouse hearts and whether phospholipase C (PLC) is involved. Rat cardiomyocytes were treated with the A(1)R agonist chlorocyclopentyladenosine (CCPA) and exposed to primary PKC-epsilon and RACK2 antibodies with secondaries conjugated to Cy3 and Cy5 (indodicarbocyanine), respectively. Scanning confocal microscopy showed that CCPA caused PKC-epsilon to reversibly colocalize with RACK2 within 3 min. Additionally, rat and mouse hearts were perfused and stimulated with CCPA or phenylisopropyladenosine to activate A(1)R, or with phorbol 12-myristate 13-acetate to activate PKC. RACK2 was immunoprecipitated from heart extracts and resolved with SDS-PAGE. Western blotting showed that CCPA, phenylisopropyladenosine, and phorbol 12-myristate 13-acetate in the rat heart increased the PKC-epsilon co-IP with RACK2 by 186, 49, and >1,000%, respectively. The A(1)R antagonist 8-cyclopentyl-1,3-dipropylxanthine prevented the CCPA-induced co-IP with RACK2. In mouse hearts, CCPA increased the co-IP of PKC-epsilon with RACK2 by 61%. With rat cardiomyocytes, the beta-adrenergic agonist isoproterenol increased sarcomere shortening by 177%. CCPA reduced this response by 47%, an action inhibited by the PLC inhibitor U-73122 and 8-cyclopentyl-1,3-dipropylxanthine. In conclusion, A(1)R stimulation of the heart is associated with PLC-initiated PKC-epsilon translocation and association with RACK2.

Adenosine A2A and beta-adrenergic calcium transient and contractile responses in rat ventricular myocytes.

The adenosine A2A receptor (A2AR) enhances cardiac contractility, and the adenosine A1R receptor (A1R) is antiadrenergic by reducing the adrenergic beta1 receptor (beta1R)-elicited increase in contractility. In this study we compared the A2AR-, A1R-, and beta1R-elicited actions on isolated rat ventricular myocytes in terms of Ca transient and contractile responses involving PKA and PKC. Stimulation of A2AR with 2 microM (approximately EC50) CGS-21680 (CGS) produced a 17-28% increase in the Ca transient ratio (CTR) and maximum velocities (Vmax) of transient ratio increase (+MVT) and recovery (-MVT) but no change in the time-to-50% recovery (TTR). CGS increased myocyte sarcomere shortening (MSS) and the maximum velocities of shortening (+MVS) and relaxation (-MVS) by 31-34% with no change in time-to-50% relengthening (TTL). beta1R stimulation using 2 nM (approximately EC50) isoproterenol (Iso) increased CTR, +MVT, and -MVT by 67-162% and decreased TTR by 43%. Iso increased MSS, +MVS, and -MVS by 153-174% and decreased TTL by 31%. The A2AR and beta1R Ca transient and contractile responses were not additive. The PKA inhibitor Rp-adenosine 3',5'-cyclic monophosphorothioate triethylamonium salt prevented both the CGS- and Iso-elicited contractile responses. The PKC inhibitors chelerythrine and KIE1-1 peptide (PKCepsilon specific) prevented the antiadrenergic action of A1R but did not influence A2AR-mediated increases in contractile variables. The findings suggest that cardiac A2AR utilize cAMP/PKA like beta1R, but the Ca transient and contractile responses are less in magnitude and not equally affected. Although PKC is important in the A1R antiadrenergic action, it does not seem to play a role in A2AR-elicited Ca transient and contractile events.

Adenosine A1 and A2A receptor regulation of protein phosphatase 2A in the murine heart.

Adenosine plays a role in regulating the contractile function of the heart. This includes a positive ionotropic action via the adenosine A(2A) receptor (A(2A)R) and an inhibition of beta(1)-adrenergic receptor-induced ionotropy (antiadrenergic action) via the adenosine A(1) receptor (A(1)R). Phosphatase activity has also been shown to influence contractile function by affecting the level of protein phosphorylation. Protein phosphatase 2A (PP2A) plays a significant role in mediating the A(1)R antiadrenergic effect. The purpose of this study was to investigate the effects of A(2A)R and A(1)R on the activities of PP2A in hearts obtained from wild-type (WT) and A(2A)R knockout (A(2A)R-KO) mice. PP2A activities were examined in myocardial particulate and cytoplasmic extract fractions. Treatment of wild-type hearts with the A(1)R agonist CCPA increased the total PP2A activity and increased the particulate:cytoplasmic PP2A activity ratio. Treatment with the A(2A)R agonist CGS-21680 (CGS) decreased the total PP2A activity and decreased the particulate:cytoplasmic PP2A activity ratio. This indicated a movement of PP2A activity between cell fractions. The effect of CCPA was inhibited by CGS. In A(2A)R-KO hearts the response to A(1)R activation was markedly enhanced whereas the response to A(2A)R activation was absent. These data show that A(2A)R and A(1)R regulate PP2A activity, thus suggesting an important mechanism for modulating myocardial contractility.

Adenosine A1 and A2A receptor effects on G-protein cycling in beta-adrenergic stimulated ventricular membranes.

In the heart beta1-adrenergic (beta1R) and adenosine A1 (A1R) and A2A (A2AR) receptors modulate contractile and metabolic function. The interaction between these receptors was investigated at the level of G-protein cycling by determining the effect of receptor agonists on the binding of GTP to G-proteins and displacement of G alpha-subunit-bound GDP by GTP. Crude membranes from rat heart or brain were stimulated by agonists for beta1R (isoproterenol; ISO), A1R (chlorocyclopentyladenosine, CCPA) and A2AR (CGS-21680; CGS). GTP binding to membranes was increased by ISO (17%), CCPA (6%) and CGS (12%). Binding values observed with incubation using ISO and CCPA together were significantly less than values obtained by the incubation of individual agents alone. With ISO, GTP binding to G alpha(s) subunits as determined by immunoprecipitation was increased 79% in heart and 87% in brain. These increases were attenuated by CCPA, an effect that was inhibited by CGS. GDP release by membranes was increased 6.9% and 4.6% by ISO and CCPA, respectively. After co-incubation of these agonists, release was increased less than determined by the addition of the individual agent responses. CGS inhibited the reduced release caused by of CCPA. Adenylyl cyclase activity stimulated by ISO was attenuated 33% by CCPA, an effect inhibited by CGS. Together, these results indicate that A1R exert an antiadrenergic action at the level of beta1R stimulated G(s)-protein cycling and that A2AR reduce this action.

Endogenous adenosine inhibits CNS terminal Ca(2+) currents and exocytosis.

Bursts of action potentials (APs) are crucial for the release of neurotransmitters from dense core granules. This has been most definitively shown for neuropeptide release in the hypothalamic neurohypophysial system (HNS). Why such bursts are necessary, however, is not well understood. Thus far, biophysical characterization of channels involved in depolarization-secretion coupling cannot completely explain this phenomenon at HNS terminals, so purinergic feedback mechanisms have been proposed. We have previously shown that ATP, acting via P2X receptors, potentiates release from HNS terminals, but that its metabolite adenosine, via A(1) receptors acting on transient Ca(2+) currents, inhibit neuropeptide secretion. We now show that endogenous adenosine levels are sufficient to cause tonic inhibition of transient Ca(2+) currents and of stimulated exocytosis in HNS terminals. Initial non-detectable adenosine levels in the static bath increased to 2.9 microM after 40 min. These terminals exhibit an inhibition (39%) of their transient inward Ca(2+) current in a static bath when compared to a constant perfusion stream. CPT, an A(1) adenosine receptor antagonist, greatly reduced this tonic inhibition. An ecto-ATPase antagonist, ARL-67156, similarly reduced tonic inhibition, but CPT had no further effect, suggesting that endogenous adenosine is due to breakdown of released ATP. Finally, stimulated capacitance changes were greatly enhanced (600%) by adding CPT to the static bath. Thus, endogenous adenosine functions at terminals in a negative-feedback mechanism and, therefore, could help terminate peptide release by bursts of APs initiated in HNS cell bodies. This could be a general mechanism for controlling transmitter release in these and other CNS terminals.

Contractile effects of adenosine A1 and A2A receptors in isolated murine hearts.

The adenosine A1 receptor (A1R) inhibits beta-adrenergic-induced contractile effects (antiadrenergic action), and the adenosine A2A receptor (A2AR) both opposes the A1R action and enhances contractility in the heart. This study investigated the A1R and A2AR function in beta-adrenergic-stimulated, isolated wild-type and A2AR knockout murine hearts. Constant flow and pressure perfused preparations were employed, and the maximal rate of left ventricular pressure (LVP) development (+dp/dt(max)) was used as an index of cardiac function. A1R activation with 2-chloro-N6-cyclopentyladenosine (CCPA) resulted in a 27% reduction in contractile response to the beta-adrenergic agonist isoproterenol (ISO). Stimulation of A2AR with 2-P(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxyamidoadenosine (CGS-21680) attenuated this antiadrenergic effect, resulting in a partial (constant flow preparation) or complete (constant pressure preparation) restoration of the ISO contractile response. These effects of A2AR were absent in knockout hearts. Up to 63% of the A2AR influence was estimated to be mediated through its inhibition of the A1R antiadrenergic effect, with the remainder being the direct contractile effect. Further experiments examined the effects of A2AR activation and associated vasodilation with low-flow ischemia in the absence of beta-adrenergic stimulation. A2AR activation reduced by 5% the depression of contractile function caused by the flow reduction and also increased contractile performance over a wide range of perfusion flows. This effect was prevented by the A2AR antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385). It is concluded that in the murine heart, A1R and A2AR modulate the response to beta-adrenergic stimulation with A2AR, attenuating the effects of A1R and also increasing contractility directly. In addition, A2AR supports myocardial contractility in a setting of low-flow ischemia.

Inhibition of phosphatase activity enhances preconditioning and limits cell death in the ischemic/reperfused aged rat heart.

Brief, nonlethal episodes of ischemia in the mammalian heart provide cardioprotection against the detrimental effects of a longer duration ischemia. The manifestation of this preconditioning (PC) phenomenon is initiated by the enhanced phosphorylation state of signal transduction proteins. We reported previously that PC is decreased in the aged rat myocardium. Although the mechanism responsible for this loss is not understood, a reduction in the phosphorylation of critical proteins associated with PC may be postulated. Experiments were conducted to investigate whether PC in the aged heart can be restored with the inhibition of endogenous protein phosphatases thereby enhancing phosphorylation of signaling proteins. Levels of phosphatase activities were also assessed with adult heart aging. Hearts from young adult (3-4 mo.) and aged (21-22 mo.) Fischer-344 rats were perfused in the presence or absence of okadaic acid (OKA; 0.1 microM). Aged adult hearts were either not preconditioned or were preconditioned with two PC cycles (5 min ischemia/5 min reperfusion). Myocardial cellular death that developed with a subsequent ischemia was determined with triphenyltetrazolium. With PC, 55% of the aged heart after ischemia was no longer viable. OKA administered before or after ischemia reduced this ischemia-induced cellular death by 29%. Without PC, OKA reduced viability 18% only when present before and after the ischemic episode. OKA in the ischemic young heart during reperfusion reduced the loss of viability 31%. The Protein Phosphatase 2A (PP2A) activity was found to be up to 82% greater in ventricular myocardium of aged rats. In conclusion, aging-induced changes in protein dephosphorylation may be one mechanism reducing the manifestation of preconditioning in the aged heart.

Protein kinase Cepsilon and the antiadrenergic action of adenosine in rat ventricular myocytes.

Adenosine-induced antiadrenergic effects in the heart are mediated by adenosine A(1) receptors (A(1)R). The role of PKCepsilon in the antiadrenergic action of adenosine was explored with adult rat ventricular myocytes in which PKCepsilon was overexpressed. Myocytes were transfected with a pEGFP-N1 vector in the presence or absence of a PKCepsilon construct and compared with normal myocytes. The extent of myocyte shortening elicited by electrical stimulation of quiescent normal and transfected myocytes was recorded with video imaging. PKCepsilon was found localized primarily in transverse tubules. The A(1)R agonist chlorocyclopentyladenosine (CCPA) at 1 microM rendered an enhanced localization of PKCepsilon in the t-tubular system. The beta-adrenergic agonist isoproterenol (Iso; 0.4 microM) elicited a 29-36% increase in myocyte shortening in all three groups. Although CCPA significantly reduced the Iso-produced increase in shortening in all three groups, the reduction caused by CCPA was greatest with PKCepsilon overexpression. The CCPA reduction of the Iso-elicited shortening was eliminated in the presence of a PKCepsilon inhibitory peptide. These results suggest that the translocation of PKCepsilon to the t-tubular system plays an important role in A(1)R-mediated antiadrenergic actions in the heart.

Receptors subtypes involved in adenosine-mediated modulation of norepinephrine release from cardiac nerve terminals.

The objective of this study was to determine which adenosine receptor subtypes were involved in the modulation of norepinephrine release from cardiac nerve terminals. In addition, the persistence of adenosine-mediated effects was evaluated. Rat hearts attached to the stellate ganglion were isolated and perfused. The ganglion was electrically stimulated twice (S1 and S2), allowing 10 min between the stimulations. To determine adenosine receptor subtypes, selective and nonselective adenosine agonists and antagonists were infused following S1 and until the end of S2. To evaluate the persistence of adenosine-mediated effect on norepinephrine release, the stellate ganglion was stimulated a third (S3) and fourth (S4) time. Coronary effluents were collected to determine norepinephrine content. Adenosine and a selective A1 receptor agonist, CCPA, inhibited norepinephrine release by 49% and 54%, respectively. This effect was reversed by simultaneous infusion of nonspecific (8-SPT) and specific (DPCPX) A1 receptor antagonists. Selective A2A (CGS 21680) and A3 (AB-MECA) receptor agonists had no discernible effect on norepinephrine release. Similarly, adenosine A2A receptor antagonists CSC and DMPX did not alter the dose-response relation between norepinephrine release and adenosine. Finally, the inhibitory effects of adenosine on norepinephrine release did not persist 10 min subsequent to the removal of adenosine. Adenosine inhibited norepinephrine release primarily via the adenosine A1 receptor. This effect of adenosine was of short duration. Adenosine A2A and A3 receptors were either absent or functionally insignificant in the regulation of norepinephrine release in the rat heart.

Molecular mechanisms of reduced beta-adrenergic signaling in the aged heart as revealed by genomic profiling.

Myocardial aging leads to a reduction of beta-adrenergic receptor-induced metabolic and contractile responsiveness. We hypothesize that a change in the patterns of gene expression is important in these age-related events. To test this, hearts were harvested from young and aged male rats (3-4 and 20-22 mo, respectively). Total mRNA was extracted and prepared for hybridization to Affymetrix U34A GeneChips. Filtering criteria, involving fold change and a statistical significance cutoff were employed, yielding 263 probe pairs exhibiting differential signals. Of the 163 annotated genes, at least 56 (34%) were classified as signaling/cell communication. Of these 56, approximately half were directly involved in G protein-coupled receptor signaling pathways. We next determined which of these changes might be involved in anti-adrenergic activity and identified 19 potentially important gene products. Importantly, we observed a decrease in beta1-adrenergic receptor and adenylyl cyclase mRNAs, whereas the mRNA encoding beta-arrestin increased. Furthermore, the results demonstrate an increase in mRNAs encoding the adenosine A1 receptor and phospholipase D, which could increase anti-adrenergic effects. Moreover, the mRNAs encoding the muscarinic M3 receptor, nicotinic acetylcholine receptor beta3, and nicotinic acetylcholine receptor-related protein were increased as was the mRNA encoding guanylate kinase-associated protein. Interestingly, we also observed eight mRNAs whose abundance changed three- to sixfold with aging that could be considered as being compensatory. Although these results do not prove causality, they demonstrate that cardiac aging is associated with changes in the profiles of gene expression and that many of these changes may contribute to reduced adrenergic signaling.

Beta-adrenergic and antiadrenergic modulation of cardiac adenylyl cyclase is influenced by phosphorylation.

Adenosine protects the myocardium of the heart by exerting an antiadrenergic action via the adenosine A1 receptor (A1R). Because beta 1-adrenergic receptor (beta 1R) stimulation elicits myocardial protein phosphorylation, the present study investigated whether protein kinase A (PKA) catalyzed rat heart ventricular membrane phosphorylation affects the beta 1R adrenergic and A1R adenosinergic actions on adenylyl cyclase activity. Membranes were either phosphorylated with PKA in the absence/presence of a protein kinase inhibitor (PKI) or dephosphorylated with alkaline phosphatase (AP) and assayed for adenylyl cyclase activity (AC) in the presence of the beta 1R agonist isoproterenol (ISO) and/or the A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA). 32P incorporation into the protein substrates of 140-120, 43, and 29 kDa with PKA increased both the ISO-elicited activation of AC by 51-54% and the A1R-mediated reduction of the ISO-induced increase in AC by 29-50%, thereby yielding a total antiadrenergic effect of approximately 78%. These effects of PKA were prevented by PKI. AP reduced the ISO-induced increase in AC and eliminated the antiadrenergic effect of CCPA. Immunoprecipitation of the solubilized membrane adenylyl cyclase with the use of a polyclonal adenylyl cyclase VI antibody indicated that the enzyme is phosphorylated by PKA. These results indicate that the cardioprotective effect of adenosine afforded by its antiadrenergic action is facilitated by cardiac membrane phosphorylation.

Anoxia-induced changes in purine nucleoside metabolism of in vitro aged human fibroblasts.

Inosine deriving from the metabolism of adenosine or inosine monophosphate (IMP) in the fibroblast provides the substrate for xanthine oxidase and is, therefore, an important source of toxic oxygen free radicals. With well-oxygenated medium, adenosine release appears to be greater for aged than young fibroblasts. In that the adenosine release by young cells is enhanced by reduced oxygenation, the effect anoxic stress on the release of the purine nucleosides adenosine and inosine by low-passage (PDL 23-26; young) vs. high-passage (PDL 43-51; aged) human lung fibroblasts (IMR-90) was studied. Cultures of confluent fibroblasts were incubated for 16 hr under normoxic (NF) or anoxic (AF) atmospheres. The release of adenosine and inosine was determined by HPLC at 0, 3, 6 and 24 hr after termination of the 16-hr period. Immediately following anoxia (time 0), adenosine release by young AF was 29% greater than for young NF, whereas both the youn