NIR spectroscopic imaging to map hemoglobin + myoglobin oxygenation, their concentration and optical pathlength across a beating pig heart during surgery.

The purpose of this paper is to demonstrate that near-infrared (NIR) spectroscopic imaging can provide spatial distribution (maps) of the absolute concentration of hemoglobin + myoglobin, oxygen saturation parameter and optical pathlength, reporting on the biochemico-physiological status of a beating heart in vivo. The method is based on processing the NIR spectroscopic images employing a first-derivative approach. Blood-pressure-controlled gating compensated the effect of heart motion on the imaging. All the maps are available simultaneously and noninvasively at a spatial resolution in the submillimeter range and can be obtained in a couple of minutes. The equipment has no mechanical contact with the tissue, thereby leaving the heart unaffected during the measurement.

Potassium fluxes, energy metabolism, and oxygenation in intact diabetic rat hearts under normal and stress conditions.

We evaluated the function of Na(+)/K(+) ATPase and sarcolemmal K(ATP) channels in diabetic rat hearts. Six weeks after streptozotocin (STZ) injection, unidirectional K(+) fluxes were assayed by using (87)rubidium ((87)Rb(+)) MRS. The hearts were loaded with Rb(+) by perfusion with Krebs-Henseleit buffer, in which 50% of K(+) was substituted with Rb(+). The rate constant of Rb(+) uptake via Na(+)/K(+) ATPase was reduced. K(ATP)-mediated Rb(+) efflux was activated metabolically with 2,4-dinitrophenol (DNP, 50 micromol.L(-1)) or pharmacologically with a K(ATP) channel opener, P-1075 (5 micromol.L(-1)). Cardiac energetics were monitored by using (31)P MRS and optical spectroscopy. DNP produced a smaller ATP decrease, yet similar Rb(+) efflux activation in STZ hearts. In K(+)-arrested hearts, P-1075 had no effect on high-energy phosphates and stimulated Rb(+) efflux by interaction with SUR2A subunit of K(ATP) channel; this stimulation was greater in STZ hearts. In normokalemic hearts, P-1075 caused cardiac arrest and ATP decline, and the stimulation of Rb(+) efflux was lower in normokalemic STZ hearts arrested by P-1075. Thus, the Rb(+)efflux stimulation in STZ hearts was altered depending on the mode of K(ATP) channel activation: pharmacologic stimulation (P-1075) was enhanced, whereas metabolic stimulation (DNP) was reduced. Both the basal concentration of phosphocreatine ([PCr]) and [PCr]/[ATP] were reduced; nevertheless, the STZ hearts were more or equally resistant to metabolic stress.

A serine kinase associates with the RAL GTPase and phosphorylates RAL-interacting protein 1.

A kinase activity that phosphorylated myelin basic protein in vitro was detected in RalA and RalB immunoprecipitates from human platelets. Protein-protein interaction studies using recombinant GST-RalA, GST-RalB and GST-cH-Ras confirmed that the kinase specifically associates with the Ral GTPase. The Ral Interacting Protein 1 (RIP1), a GTPase Activating Protein (GAP) for Cdc42 and Rac1, was found to be the preferred substrate for the Ral Interacting Kinase (RIK). Phosphoamino acid analysis demonstrated that RIK phosphorylated serine residue in RIP1. The Ral-RIK interaction was not dependent on the guanine nucleotide status of Ral. RIK was detected in a variety of rat tissues with testis containing the highest and skeletal muscle the lowest activity. In-gel kinase renaturation assay using RIP1 as the substrate demonstrated that the kinase activity was associated with polypeptides of molecular mass of approximately 36-40 kDa and was detected in most rat tissues with a prominent 38 kDa band in testis and a 40 kDa band in brain. Human platelets contained a single band of approximately 36 kDa. RIK was distinct from MAPKs, CDKs, cyclic AMP dependent protein kinase and Ca2+/calmodulin dependent kinases. To demonstrate in vivo interaction, the endogenous Ral-RIK complex was isolated using a calmodulin affinity column. The Ral-RIK complex co-eluted from this column upon washing with a 13 residue peptide that encompasses the calmodulin-binding domain in RalA. The data suggest that RIK is a serine specific kinase that phosphorylates RIP1 and is constitutively associated with Ral. The current study provides additional support for a link between Ral and the Cdc42/Rac1 signalling pathways in the cell.

Potassium transport in Langendorff-perfused mouse hearts assessed by 87Rb NMR spectroscopy.

We studied the fluxes of a potassium congener (Rb(+)) in mouse hearts by (87)Rb MRS at 8.4T. The hearts were loaded with Rb(+) by perfusion with Krebs-Henseleit buffer, in which 50% of K(+) was substituted with Rb(+). We initiated Rb(+) efflux by changing the perfusion medium to Rb(+)-free buffer. Spectra were acquired every 1.85 min, and the kinetics of Rb(+) transport were analyzed by means of monoexponential fits. The rate constants of Rb(+) uptake and efflux were 0.0680 +/- 0.0028 and 0.0510 +/- 0.0051 min(-1), respectively (approximately 30% faster than in the rat heart). The ATP-sensitive potassium channel opener, P-1075 (5 microM), and mitochondrial uncoupler, 2,4-dintrophenol (50 microM), activated Rb(+) efflux from mouse hearts by approximately 35%. The mechanisms responsible for the differences in Rb(+) uptake and efflux under baseline conditions and stimulation, in comparison with rat hearts, are discussed. These data provide a background for studies of cardiac potassium transport in transgenic mouse strains.

Sarcolemmal and mitochondrial effects of a KATP opener, P-1075, in "polarized" and "depolarized" Langendorff-perfused rat hearts.

We investigated consequences of cardiac arrest on sarcolemmal and mitochondrial effects of ATP-sensitive potassium channel (KATP) opener, P-1075, in Langendorff-perfused rat hearts. Depolarised cardiac arrest (24.7 mM KCl) did not affect glibenclamide-sensitive twofold activation of rubidium efflux by P-1075 (5 microM) from rubidium-loaded hearts, but eliminated uncoupling produced by P-1075 in beating hearts: 40% depletion of phosphocreatine and ATP, 50% increase in oxygen consumption, and reduction of cytochrome c oxidase. Depolarized cardiac arrest by calcium channel blocker, verapamil (5 microM), also prevented uncoupling. Lack of P-1075 mitochondrial effects in depolarized hearts was not due to changes in phosphorylation potential, because 2,4-dintrophenol (10 microM) reversed the [PCr]/[Cr] increase and Pi decrease, characteristic of KCl-arrest, but did not restore uncoupling. In agreement with this conclusion, pyruvate (5 mM) increased [PCr]/[Cr] and decreased Pi, but did not prevent uncoupling in beating hearts. A decrease in mean [Ca2+] in KCl-arrested hearts could not account for lack of P-1075 mitochondrial effects, because calcium channel opener, S-(-)-Bay K8644 (50 nM), and beta-agonist, isoproterenol (0.5 microM), did not facilitate uncoupling. In contrast, in adenosine (1 mM)-arrested hearts (polarized arrest), P-1075 caused 40% phosphocreatine and ATP depletion. In isolated rat liver mitochondria, P-1075 (20 microM) decreased mitochondrial membrane potential (DeltaPsi) by approximately 14 mV (demonstrated by redistribution of DeltaPsi-sensitive dye, rhodamine 800) in a glibenclamide-sensitive manner. We concluded that cell membrane depolarization does not prevent activation of sarcolemmal KATP by P-1075, but it plays a role in mitochondrial uncoupling effects of P-1075.

Cardioselective sulfonylthiourea HMR 1098 blocks mitochondrial uncoupling induced by a KATP channel opener, P-1075, in beating rat hearts.

We investigated effects of blockade of cardiac ATP-sensitive potassium channels (KATP) with a novel cardioselective sulfonylthiourea, HMR 1098, on metabolic uncoupling caused by a potent KATP opener, P-1075, in Langendorff-perfused rat hearts. We used (1) 87Rb-NMR to detect activation-deactivation of sarcolemmal KATP, (2) 31P-NMR to monitor high-energy phosphates, (3) oxygen uptake measurements to monitor cellular respiration, and (4) myocardial optical absorbance measurements at 603 nm to follow changes in cytochrome c oxidase redox state. Activation of sarcolemmal KATP by P-1075 (5 microM) and a mitochondrial uncoupler 2,4-dinitrophenol (DNP) (50 microM) stimulated Rb+ efflux from the hearts by 130% and 60%, respectively. HMR 1098 (5 and 30 microM) blocked activation of sarcolemmal KATP in situ. HMR 1098 also prevented cardiac arrest and mitochondrial uncoupling induced by P-1075, such as (a) depletion of phosphocreatine and ATP by 40%, (b) two-fold decrease in venous oxygen, and (c) reduction of cytochrome c oxidase (demonstrated by an increase in 603 nm optical absorbance). The metabolic effects of P-1075 can be readily explained by activation of putative mitochondrial KATP. We concluded that blockade of mitochondrial uncoupling by HMR 1098 included an inhibiting effect of HMR 1098 on sarcolemmal and mitochondrial KATP in beating rat hearts.

Effects of K(ATP) channel openers, P-1075, pinacidil, and diazoxide, on energetics and contractile function in isolated rat hearts.

We investigated the metabolic effects of a potent opener of ATP-sensitive K(+) (K(ATP)) channels, P-1075, in perfused rat hearts with the help of(31)P NMR spectroscopy. A 20 min infusion of 5 microm P-1075 depleted phosphocreatine and ATP by approximately 40%, concomitantly with a two-fold increase in inorganic phosphate, while oxygen consumption by the hearts increased by 50%. P-1075 induced a cardiac contracture (left ventricular end diastolic pressure increased from 6 to 60 mmHg) and a cardiac arrest after an infusion of approximately 9 min. The effects were fully reversed by glibenclamide (5 microm), but not by sodium 5-hydroxydecanoate (0.4 m m). A P-1075-related K(ATP) opener, pinacidil (0.3 m m), partially reversed the effects of P-1075, but a structurally unrelated opener, diazoxide (0.5 m m), had no effect. Pinacidil and diazoxide alone did not significantly affect PCr and ATP levels. Bioenergetic and functional effects similar to those of P-1075 were induced by infusion of a classic mitochondrial uncoupler, 2,4-dinitrophenol (50 microm); however, they were not abolished by glibenclamide. In addition, it was shown, using(87)Rb NMR, that both agents, P-1075 and 2,4-dinitrophenol, resulted in a stimulation of Rb(+) efflux from the Rb(+) loaded rat hearts by approximately 130 and 65%, respectively, in a glibenclamide-sensitive manner. An inhibitory effect of P-1075 on ATP synthesis cannot be explained by its well-known action on sarcolemmal K(ATP) channels. We concluded that, unlike an uncoupling effect of 2,4-dinitrophenol, an inhibitory effect of P-1075 is produced by uncoupling of oxidative phosphorylation through the activation of mitochondrial K(ATP) channels.