Timothy P. Geisbuhler

Adenine nucleotide metabolism and compartmentalization in isolated adult rat heart cells.

The metabolism and intracellular compartmentalization of adenine nucleotides in a preparation of adult rat heart myocytes showing good morphology, viability, and tolerance to calcium ion has been examined by high performance liquid chromatography. These myocytes contain an average of 23 nmol adenine nucleotide per milligram protein which is about 60% of the adenine nucleotide content of intact rat heart tissue. The loss of adenine nucleotide occurs during the incubation and washing steps that increase the yield of viable cells, rather than during the collagenase perfusion. An analysis of cellular compartments shows that the adenine nucleotide of the cell consists of 17 nmol adenine nucleotide in the cytosol, 5 nmol in the mitochondria, and 1.3 nmol adenosine diphosphate bound to myofibrils per milligram cell protein. Myocytes lose both adenosine triphosphate and adenine nucleotide when incubated anaerobically in the absence of glucose, and the lost adenine nucleotide can be accounted for as increased inosine, adenosine, and inosine monophosphate. Myocytes that contain less than 0.1 nmol of cytosol adenosine triphosphate per milligram cell protein maintain an intact sarcolemma, but are unable to carry out anaerobic glycolysis. Reoxygenation of anaerobic cells results in restoration of energy charge and a net resynthesis of about 2 nmol adenine nucleotide per milligram protein. Adenosine and inosine monophosphate decrease on reaerarion of anaerobic cells, whereas inosine levels increase. When iodoacetate is added to block glycolysis, the decline in adenine nucleotide and production of inosine monophosphate are accelerated and there is no resynthesis of adenine nucleotide when anaerobic cells are reoxygenated. Large accumulations of inosine monophosphate are also seen in myocytes treated with an uncoupler of oxidative phosphorylation.

Ruthenium red-sensitive and -insensitive release of Ca2+ from uncoupled heart mitochondria.

The uncoupler-induced release of accumulated Ca2+ from heart mitochondria can be separated into two components, one sensitive and one insensitive to ruthenium red. In mitochondria maintaining reduced NAD(P)H pools and adequate levels of endogenous adenine nucleotides, the release of Ca2+ following addition of an uncoupler is virtually all inhibited by ruthenium red and can be presumed to occur via reversal of the Ca2+ uniporter. When ruthenium red is added to block efflux via this pathway, high rates of Ca2+ efflux can still be induced by an uncoupler, provided either NADH is oxidized or mitochondrial adenine nucleotide pools are depleted by prior treatment. This ruthenium red-insensitive Ca2+-efflux pathway is dependent on the level of Ca2+ accumulated and is accompanied by swelling of the mitochondria and loss of endogenous Mg2+. Loss of Ca2+ by this relatively nonspecific pathway is strongly inhibited by Sr2+ and by nupercaine, as well as by oligomycin and exogenous adenine nucleotides. The loss of Ca2+ from uncoupled heart mitochondria occurs via a combination of these two mechanisms except under conditions chosen specifically to limit efflux to one or the other pathway.

Guanosine metabolism in adult rat cardiac myocytes: ribose-enhanced GTP synthesis from extracellular guanosine

The metabolic fate of transported guanosine was examined in adult rat cardiac myocytes. Freshly isolated cells were incubated with 10 μM or 100 μM [3H]guanosine and the nucleotide products extracted and examined for radiolabel distribution. The data presented show significant incorporation of guanosine into the 5′-nucleotide pool, and a marked stimulation of that incorporation by ribose. An average of 233 pmol/mg cell protein extracellular guanosine was incorporated into the cellular 5′-nucleotides over 90 min at both 10 μM and 100 μM external nucleoside. This appeared primarily as GTP (approx. 204 pmol/mg cell protein in 90 min). Only guanine nucleotides contained radiolabel; adenine nucleotides and IMP remained unlabelled even after 90 min incubation of the cells with [3H]guanosine. Addition of 5 mM ribose to the medium stimulated guanosine incorporation into 5′-nucleotides 1.6-fold (380 pmol/mg protein vs 234 pmol/mg over 90 min at 10 μM guanosine), but did not enhance the amount of guanosine transported into the cells. Intracellular guanosine concentrations exceeded those of the incubation medium at both external guanosine concentrations studied. More [3H]guanosine was salvaged at 100 μM than at 10 μM external guanosine (562 vs 380 pmol/mg protein in 90 min), but only if ribose was present in the medium. We conclude from these studies that guanosine is salvaged by heart muscle, and that at high guanosine levels the rate of guanosine salvage appears dependent on the availability of phosphoribosylpyrophosphate within the cells. At lower guanosine levels in the presence of ribose, cell guanine concentrations limit the rate of guanosine incorporation into 5′-nucleotides.

Forskolin inhibition of hexose transport in cardiomyocytes

The effects of insulin, forskolin, isoproterenol, and epinephrine on 3-O-methylglucose (hexose) transport and cell cyclic AMP levels were determined in adult rat cardiomyocytes. Insulin stimulated hexose transport in these cells an average of 2.5-fold. Initial hexose transport rates at 1 mM hexose were 3.75×10−2 nmol/mg cell protein/second in the absence of insulin, and 8.25×10−2 nmol/mg cell protein/second in the presence of 12.3 μM insulin. Forskolin at 5 μM nearly abolished hexose transport within 3 s of exposure, but did not increase cell cyclic AMP concentrations within 9 s. The apparentKi for hexose transport inhibition was about 0.3 μM forskolin. Epinephrine and isoproterenol at 50 μM increased cell cyclic AMP 4-fold during 9 s exposure, but did not affect hexose transport. Treatment of cells with these catecholamines of forskolin for up to 99 s increased cell cyclic AMP, but only forskolin inhibited hexose transport. We coclude from these results that forskolin acts on hexose transport independent of its action on adenyl cyclase, and that cyclic AMP does not inhibit or stimulate hexose transport.

Cardioprotective Effect of Hydroxysafflor Yellow A via the Cardiac Permeability Transition Pore

Myocardial ischemia damages cardiac myocytes in part via opening of the mitochondrial permeability transition pore. Preventing this pores opening is therefore a useful therapeutic goal in treating cardiovascular disease. Hydroxysafflor yellow A has been proposed as a nontoxic alternative to other agents that modulate mitochondrial permeability transition pore opening. In this study, we proposed that hydroxysafflor yellow A prevents mitochondrial permeability transition pore formation in anoxic cardiac myocytes, and thus protects the cell from damage seen during reoxygenation of the cardiac myocytes. Experiments with hydroxysafflor yellow A transport in aerobic myocytes show that roughly 50% of the extracellular dye concentration crosses the cell membrane in a 2-h incubation. In our anoxia/reoxygenation protocol, hydroxysafflor yellow A modulated both the reduction of viability and the loss of rod-shaped cells that attend anoxia and reoxygenation. Hydroxysafflor yellow As protective effect was similar to that of cyclosporin A, an agent known to inhibit mitochondrial permeability transition pore opening. In additional experiments, plated myocytes were loaded with calcein/MitoTracker Red, then examined for intracellular dye distribution/morphology after anoxia/reoxygenation. Hydroxysafflor yellow A-containing cells showed a cardioprotective pattern similar to that of cyclosporin A (an agent known to close the mitochondrial permeability transition pore). We conclude that hydroxysafflor yellow A can enter the cardiac myocyte and is able to modulate anoxia/reoxygenation-induced damage by interacting with the mitochondrial permeability transition pore.

The Effect of Hawthorn Extract on Coronary Flow

Hawthorn extract has been used for heart failure and may decrease cardiac cell injury and improve cardiac function. One proposed mechanism for hawthorn action is vasodilation. We hypothesized that hawthorn extract would increase coronary blood flow in isolated perfused rat hearts. Coronary flow was measured in nonworking perfused rat hearts (Langendorff, constant pressure) using a flow probe; data were collected electronically in real time. Hawthorn extract showed an early (30-120 seconds) vasodilation, followed by a later (3-5 minutes) decrease in coronary flow. Maximum vasodilation occurred with 240 μg/mL hawthorn extract. Hawthorn’s pattern of activity was unlike that of several known vasoactive drugs. Both nitric oxide synthase inhibitors and indomethacin abolished early vasodilation, but they had no effect on the late phase decrease in flow. We suggest that a hawthorn-induced increase in nitric oxide generation leads to an increase in prostacyclin production, thus causing early phase vasodilation.

Compartmentalization of non-adenine nucleotides in anoxic cardiac myocytes

Loss of 5′-nucleotides from cardiac myocytes is a distinguishing feature of myocardial ischemia. Previous work has documented dislocations of metabolic processes mediated by both purine and pyrimidine nucleotides, especially the adenine nucleotides. This study was designed to establish the extent of anoxia-induced depletion of non-adenine nucleotides in the cytosolic compartment of heart muscle cells. Cardiac myocytes were incubated aerobically (O2) or anoxically (N2) for 30 or 60 min; anoxic cells at both time points were reoxygenated for 10 min. Roughly 85–90% of cytosine triphosphate (CTP) and uridine triphosphate (UTP) were cytosolic under aerobic conditions, compared with 62% of guanosine triphosphate (GTP) and 90% of adenosine triphosphate (ATP) under similar conditions. Similarly, the total cytidine and uridine nucleotide pool of aerobic myocytes was 70–90% cytosolic vs. 61% of total guanine nucleotides and 78% of total adenine nucleotides. After the onset of anoxia, cytosolic nucleotides (principally the triphosphate forms) were quickly degraded. Reoxygenation of anoxic myocytes for 10 min allowed some recovery of ATP, GTP, and CTP, but very little recovery of UTP. The recovered nucleotide appeared almost exclusively in the cytosol. These results support the concept that non-adenine nucleotides could reach critically low levels in anoxic or ischemic heart in advance of adenine nucleotides. The importance of the depletion of non-adenine nucleotides is discussed in terms of the energetic needs of the myocyte, and the need for the cell to drive G-protein-coupled reactions, lipid synthesis, and glycogenesis.

Inhibition of citrulline synthesis by octanoate and its modulation by adenine nucleotides

Liver mitochondria from octanoate-treated rabbits showed an impaired ability to synthesize citrulline. Two methods were used to evaluate citrulline synthesis in rat liver mitochondria. Under these conditions octanoate inhibited citrulline synthesis by over 50%. When ATP was included in the assay medium the inhibitory effect of octanoate was prevented. In the absence of ATP in the suspending medium, octanoate did not significantly lower total adenine nucleotides in rat liver mitochondria. However, under these conditions octanoate caused a change in the adenine nucleotide profile such that ATP content was decreased and AMP content was increased. When ATP was present in the assay medium, octanoate caused a similar increase in AMP content. However, ATP decreased only slightly. The alterations in mitochondrial adenine nucleotide profile by octanoate and the reversal of the effect by exogenous ATP suggests that octanoate inhibits citrulline synthesis via reduced intramitochondrial ATP levels. The ability of octanoate to lower mitochondrial ATP and elevate mitochondrial AMP may be related to its intramitochondrial activation by the medium chain fatty acid activating enzyme.

Anoxia inhibits guanosine salvage in cardiac myocytes

The adult heart depends largely on salvage synthesis to supply its 5′-nucleotide needs. Previous work from this laboratory established that guanosine is metabolized into guanine 5′-nucleotides in heart cells, but that salvage rates are very slow as compared to adenosine. The author hypothesized that guanosine salvage is regulated according to the needs of the cell for guanine nucleotides. This hypothesis was tested using cardiac myocytes which were rendered anoxic for 0–60 min. During this anoxic period, guanine nucleotides were depleted about 50%. At 0, 30, and 60 min, aliquots were removed for cell counting and nucleotide analysis; 50 μM3H-guanosine was then added and the incubation continued for 1 min. The cells were then extracted and assayed for radioactivity in the guanine nucleotide products. Anoxia for 60 min, depressed GTP levels by 89%, total guanine nucleotides by 50%, and short-term guanosine salvage by 48% over aerobic controls. Reoxygenation of the myocytes after 30 min of anoxia returned guanosine salvage rates to nearly normal (87% of control). Preincubation of the myocytes with 5 mM ribose for times up to 1 hour modestly increased salvage rates of guanosine in aerobic cells. These results suggest that guanosine salvage in cardiac myocytes is not regulated by the size of the guanine nucleotide pool (that is, not sensitive to the demand for guanine nucleotides). Instead, salvage of guanosine is probably limited by cytosolic levels of ATP or phosphoribosylpyrophosphate, the production of which are dependent on adequate oxygen supplies.