1. Pathways of Rb+ Influx and Their Relation to Intracellular [Na+] in the Perfused Rat Heart
by Valerie V. Kupriyanov, Laura C. Stewart, Bo Xiang, June Kwak, and Roxanne Deslauriers
Circulation Research
Volume 76(5):
May 1, 1995
Copyright © American Heart Association, Inc. All rights reserved.

2. Left, Representative 87Rb nuclear magnetic resonance spectra of perfused rat heart during Rb+ load and washout.
Left, Representative 87Rb nuclear magnetic resonance spectra of perfused rat heart during Rb+ load and washout. Each spectrum represents sum of 7800 acquisitions collected during 2 minutes. Line broadening, 150 Hz. In this experiment, heart was fully immersed in the perfusate (bath). To reduce the signal from Rb+ in the bath, the heart was perfused from outside at a flow about three times that of the coronary flow. Other details are described in “Materials and Methods.” Right, Graph showing kinetics of Rb+ accumulation and washout in perfused rat hearts. Means±SD for five hearts are given. Rb+ content was calculated as follows: the ratio of heart peak area to the reference peak area was multiplied by the Rb+ content in the reference and divided by dry heart weight. The first two points of the kinetic curve (0 to 2 and 2 to 4 minutes) were omitted because they correspond to equilibration of Rb+ in the extracellular compartments. Extrapolation of the initial linear part of the kinetic curve to “zero” time gives the approximate amount of the Rb+ in the extracellular space and in the bath surrounding heart (≈3 μmol/g dry wt). Rb+ loading and washout was done as described in “Materials and Methods.”
Valerie V. Kupriyanov et al. Circ Res. 1995;76:
Copyright © American Heart Association, Inc. All rights reserved.

3. Left, Graph showing kinetics of Rb+ equilibration in extracellular space.
Left, Graph showing kinetics of Rb+ equilibration in extracellular space. Hearts (n=5) perfused in a “dry” mode were equilibrated with Rb+ for 30 minutes under standard conditions (0.94 mmol/L RbCl+3.76 mmol/L KCl). The [K+]+[Rb+] in the perfusate was then increased fivefold for 10 minutes and then returned to normal level. In control experiments (n=3), instead of K++Rb+ mixture, an equal amount of choline chloride (ChCl) was infused to bring its concentration to 18.8 mmol/L. The beginning and end of the infusion are indicated by arrows. Right, Graph showing relation between total Rb+ and Na+ contents and perfusion pressure in perfused rat hearts. Hearts (n=3) perfused in a “dry” mode were equilibrated with Rb+ for 30 minutes under standard conditions (0.94 mmol/L RbCl+3.76 mmol/L KCl). The perfusion pressure was then altered by changing the coronary flow, and 87Rb and 23Na NMR spectra were acquired as described in “Materials and Methods” and “Results.” Additional 87Rb nuclear magnetic resonance data were also taken from experiments (n=3) in which hearts equilibrated with Rb+ and tested with ChCl were subsequently subjected to changes in perfusion pressure as described in “Materials and Methods” and “Results.” The data are expressed as percentages of the values measured for each heart at minimal perfusion pressures (25 to 50 mm Hg). The correlations obtained by the least-squares method are described by the following equations: sodium, y= x (R2=.82) and rubidium, y= x (R2=.016).
Valerie V. Kupriyanov et al. Circ Res. 1995;76:
Copyright © American Heart Association, Inc. All rights reserved.

4. Bar graph showing effects of procaine (Proc), ouabain (Ouab), bumetanide (Bum), and 4-aminopyridine (4-AP) on the Rb+ uptake rate in perfused rat hearts.
Bar graph showing effects of procaine (Proc), ouabain (Ouab), bumetanide (Bum), and 4-aminopyridine (4-AP) on the Rb+ uptake rate in perfused rat hearts. The experiments with Proc and Proc+Ouab were performed according to protocol 2, in which the Rb+ loading and washout cycle was repeated three times in each heart in the following sequence: control→ Proc→Proc+Ouab (see “Materials and Methods”). The effects of 4-AP and Bum were tested according to basic protocol 1, in which the control cycle of Rb+ loading and washout was followed by the cycle in the presence of drug. The drugs were infused as described in “Materials and Methods.” Ouab was added directly to the perfusate to a concentration of 0.6 mmol/L. Data are presented as mean±SD.
Valerie V. Kupriyanov et al. Circ Res. 1995;76:
Copyright © American Heart Association, Inc. All rights reserved.

5. Bar graph showing effects of lidocaine (Lido), dimethylamiloride (DMA), and monensin (Mon) on the Rb+ influx rate in perfused rat hearts.
Bar graph showing effects of lidocaine (Lido), dimethylamiloride (DMA), and monensin (Mon) on the Rb+ influx rate in perfused rat hearts. The experiments were performed according to basic protocol 1, in which the control cycle of Rb+ loading and washout was followed by the cycle in the presence of drug. The drugs were infused as described in “Materials and Methods.” Data are presented as mean±SD.
Valerie V. Kupriyanov et al. Circ Res. 1995;76:
Copyright © American Heart Association, Inc. All rights reserved.

6. Bar graph showing inhibition of the Rb+ uptake rate in perfused rat hearts with dimethylamiloride (DMA) and bumetanide (Bum) in the presence of lidocaine (Lido).
Bar graph showing inhibition of the Rb+ uptake rate in perfused rat hearts with dimethylamiloride (DMA) and bumetanide (Bum) in the presence of lidocaine (Lido). The data for Rb transport were pooled for two experimental protocols, 1 and 3: (1) control→Lido→Lido/DMA and (2) Lido→Lido/drug 1→Lido/drug 2, where drug 1 and drug 2 are either DMA or Bum. The drugs were infused as described in “Materials and Methods.” Means±SD are presented. Data are expressed as a percentage of values obtained in the presence of lidocaine alone (1.10±0.30 μmol · min−1 · g dry wt−1).
Valerie V. Kupriyanov et al. Circ Res. 1995;76:
Copyright © American Heart Association, Inc. All rights reserved.

7. Changes in the 23Na spectra of perfused rat hearts caused by procaine, ouabain, and monensin.
Changes in the 23Na spectra of perfused rat hearts caused by procaine, ouabain, and monensin. The experiments with procaine and procaine + ouabain were performed according to protocol 2, and those with monensin according to basic protocol 1 (see “Materials and Methods” and legends to Figs 3 and 4). Spectra represent the changes in intracellular Na+ peak intensity induced by 10 to 15 minutes of exposure to 5 mmol/L procaine, 0.6 mmol/L ouabain in the presence of procaine, or 1 μmol/L monensin alone. The spectra represent sum of 480 acquisitions collected during 4 minutes. Gaussian multiplication was used (line broadening=−30 Hz, GB parameter=0.15) to improve resolution of the intracellular and extracellular Na+ peaks (≈0.5 and 2.5 ppm, respectively). The chemical shifts are given relative to that of the Na+ peak (not shown) measured before perfusion with the shift reagent.
Valerie V. Kupriyanov et al. Circ Res. 1995;76:
Copyright © American Heart Association, Inc. All rights reserved.

8. Bar graph showing effect of drugs that alter Rb+ and Na+ transport on intracellular Na+ content in perfused rat hearts.
Bar graph showing effect of drugs that alter Rb+ and Na+ transport on intracellular Na+ content in perfused rat hearts. The experiments with procaine (Proc) and Proc+ouabain (Ouab) were performed according to protocol 2 under constant pressure perfusion. For details see “Materials and Methods” and the legend to Table 1. The experiments with monensin (Mon, 1 μmol/L), 4-aminopyridine (4-AP, 1 mmol/L), dimethylamiloride (DMA, 15 μmol/L), and bumetanide (Bum, 30 μmol/L) were carried out according to protocol 1 under both constant-pressure (80 mm Hg) and constant-flow (16 to 22 mL/min) perfusion. The shift reagent, 5 mmol/L Dy(TTHA)3−, was present in the perfusate, and 23Na spectra were constantly acquired throughout each of the protocols. The Rb+ loading and washout procedure was used in experiments with Proc, Proc+Ouab, 4-AP, and Mon, whereas the experiments with Bum and DMA were done without Rb+. The periods of drug perfusion (12 to 15 minutes) and washout (20 minutes) were the same in both sets of experiments. Means±SD are given for three hearts in each group.
Valerie V. Kupriyanov et al. Circ Res. 1995;76:
Copyright © American Heart Association, Inc. All rights reserved.

9. Graph showing correlation between the Rb+ influx rate and the intracellular Na+ content in perfused rat hearts.
Graph showing correlation between the Rb+ influx rate and the intracellular Na+ content in perfused rat hearts. Data are taken from Figs 3, 4, and 7 and expressed as percentages of control (CON) values. Details of the experimental protocols are given in “Materials and Methods” and in the legends to the respective figures. 4-AP indicates 4-aminopyridine; Bum, bumetanide; DMA, dimethylamiloride; Mon, monensin; Proc, procaine; and Ouab, ouabain.
Valerie V. Kupriyanov et al. Circ Res. 1995;76:
Copyright © American Heart Association, Inc. All rights reserved.