NMR characteristics of intracellular K in the rat salivary gland: a 39K NMR study using double-quantum filtering

Intracellular K of the perfused rat mandibular salivary gland was measured by 39K NMR spectroscopy at 8.45 T. Multiple-quantum NMR arising from multiple-exponential decay was used to eliminate the resonance due to extracellular K in the perfused gland at 25 degrees C. The resonance due to intracellular K consisted of two Lorentzian signals stemming from the [spin 1/2 to -1/2] coherence (sharp resonance) and the [spin -1/2 to -3/2], [spin 3/2 to 1/2] coherences (broad resonance). The transverse relaxation time (T2) corresponding to the [spin 1/2 to -1/2] coherence was ca. 2.5 ms, and that corresponding to the [spin -1/2 to -3/2], [spin 3/2 to 1/2] coherences was ca. 0.4 ms. The relaxation time of the double-quantum coherence of rank 3 (originating from product operators like Ix2Iz) was determined to be ca. 0.2 ms. These results suggest the possibility of the presence of a single homogeneous population of intracellular K with a correlation time of ca. 2.5 x 10(-8) s and a quadrupolar coupling constant of ca. 1.4 MHz.     

Pulsed nuclear magnetic resonance study of 39K in frog striated muscle.

Samples of 1 M KCl solution and 10 samples of intact frog striated muscle were studied at 4-7 degrees C and/or at 21-22 degrees C. Field inhomogeneity was minimized by using small sample volumes and by using a superconducting magnet designed specifically to provide highly homogeneous fields. In the present experiments, magnetic field inhomogeneity was measured to contribute less than 15% to the free induction decay observed for intracellular 39K. The signal-to-noise ratio of the measurements was enhanced by means of extensive time-averaging. The rates of nuclear relaxation for 39K in aqueous solution were 22 +/- 3 (mean +/- 95% confidence limits) s-1 at 4-7 degrees C and 15 +/- 2 s-1 at 21-22 degrees C. For intracellular 39K, (1/T2) was measured to be 327 +/- 22 s-1 and 229 +/- 10 s-1 at the lower and higher temperatures, respectively. The corresponding values for (1/T1) in the same muscle samples were 198 +/- 31 s-1 and 79 +/- 15 s-1 at 4-7 degrees C and at 21-22 degrees C, respectively. These results for 39K are similar to those previously obtained for intracellular 23Na. Since less than 1% of the intracellular 23Na has been estimated to be immobilized, fractional immobilization of intracellular 39K is also likely to be insubstantial.     

23Na, 19F, 35Cl and 31P multinuclear nuclear magnetic resonance studies of perfused rat kidney

The concentration of intracellular sodium [Na+]i has been measured in the perfused rat kidney using 23Na nuclear magnetic resonance (NMR) in combination with the extracellular shift reagent Dy(PPPi)7-(2). The data show 100% visibility of Na+ in interstitial spaces. A measurement of the resonance intensities of intra- and extracellular 23Na ions along with a knowledge of the extracellular space as a fraction of the total kidney water space yielded an average [Na+]i of 27 +/- 2 mM for the kidney at 37 degrees C. After prolonged ischemia [Na+]i rose to approach that in the external medium. In the absence of 5% albumin in the perfusion medium, the linewidth of the 35Cl resonance of an adult kidney (45 Hz) was about twofold larger than that of the medium alone (25 Hz). In contrast, the linewidth of 35Cl resonance of an adult kidney perfused with an albumin-containing medium (82 Hz) was only about 27% of that from the medium alone (300 Hz). We interpret this effect to be due to compartmentation of albumin in the extracellular space such that the interstitial space is not freely accessible to albumin. However, for a developing, immature kidney from a growing animal, perfused with an albumin-containing medium, the linewidth of the 35Cl resonance (233 Hz) was only slightly less than that of the medium alone (300 Hz), indicating a much greater albumin permeability of the capillary walls. 19F NMR of a perfused adult kidney, loaded with the membrane-impermeant intracellular calcium indicator 5FBAPTA, yielded a value of 256 nM for [Ca2+]i. Induction of ischemia for 10 min caused the [Ca2+]i to rapidly rise to 660 nM, which could not be fully reversed by reperfusion, suggesting irreversible injury.     

Multinuclear NMR studies of the Langendorff perfused rat heart

The quantitation of intracellular sodium ion concentration [Na+]in perfused organs using NMR spectroscopy requires a knowledge of the extent of visibility of the 23Na resonance and of the intracellular volume of the organ. We have used a multinuclear NMR approach, in combination with the extracellular shift reagent dysprosium (III) tripolyphosphate, to determine the NMR visibility of intra- and extracellular 23Na and 35Cl ions, intracellular volume, and [Na+]in in the isolated Langendorff perfused rat heart. Based on a comparison of the extracellular volumes calculated using 2H and 23Na, 35Cl, or 59Co NMR of the perfused heart we conclude that resonances of extracellular sodium and chloride ions (including ions in interstitial spaces) are fully visible, contrary to assumptions in the literature. Furthermore, prolonged hypoxia or ischemia caused a dramatic increase in intracellular Na+ and [Na+] in rose to approach that in the external medium indicating full visibility of the intracellular 23Na resonance. Resonance intensities of intra- and extracellular 23Na ions, along with a knowledge of the extracellular space as a fraction of the total organ water space, yielded an average [Na+] in of about 10 mM (10 +/- 1.5 mM) for the rat heart at 37 degrees C. Double-quantum filtered 23Na NMR of the perfused rat heart in the absence and presence of paramagnetic reagents revealed, contrary to assumptions in the literature, that both intra- and extracellular sodium ions contribute to the detected signal.     

NMR measurement of cytosolic free calcium, free magnesium, and intracellular sodium in the aorta of the normal and spontaneously hypertensive rat

We have utilized multinuclear NMR spectroscopy to examine the relationship between cytosolic free Ca2+ ([Ca2+]in), free Mg2+ ([Mg2+]in) and intracellular Na+ ([Na+]in) levels of the intact thoracic aorta and primary hypertension using the Wistar-Kyoto and Sprague-Dawley rats as controls and the spontaneously hypertensive rat as a model for genetic hypertension. Cytosolic free [Ca2+] was measured using 19F NMR of the intracellular Ca2+ indicator 5,5'-difluoro-1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, free [Mg2+] using the 31P resonances of intracellular ATP, and intracellular [Na+] by 23Na NMR in combination with the extracellular shift reagent dysprosium tripolyphosphate. We have found that both the [Na+]in and [Ca2+]in levels were significantly increased in the hypertensive animals relative to normotensive controls (p less than 0.01). Mean systolic blood pressures (using tail cuff method) of control and hypertensive rats were 123 +/- 8 mm Hg (mean +/- 2 S.E., n = 7) and 159 +/- 6 mm Hg (mean +/- 2 S.E., n = 7), respectively. [Na+]in and [Ca2+]in were 21.9 +/- 6.4 mM (mean +/- 2 S.E., n = 7) and 277 +/- 28 nM (mean +/- 2 S.E., n = 5) for the spontaneously hypertensive rats versus 10.1 +/- 1.8 mM (mean +/- 2 S.E., n = 7) and 151 +/- 26 nM (mean +/- 2 S.E., n = 5) for control rats, respectively. A slight difference observed between intracellular free Mg2+ levels in hypertensives (180 +/- 38 microM, mean +/- 2 S.E., n = 4) and controls (246 +/- 76 microM, mean +/- 2 S.E., n = 4) was not statistically significant (p greater than 0.1). These data indicate alterations in the cell membrane ion transport function of the aortic smooth muscle in primary hypertension.     

Effect of monovalent cations on the pre-steady-state kinetic parameters of the plasma protease bovine activated protein C

Activated bovine plasma protein C (APC) was not reactive with the substrate p-nitrophenyl p-guanidinobenzoate (NPGB) in the absence of cations. In the presence of increasing concentrations of Na+, the acylation rate constant, k2,app, at 7 degrees C, progressively increased from 0.32 +/- 0.03 s-1 at 12.5 mM Na+ to 1.15 +/- 0.10 s-1 at 62.5 mM Na+. A linear dependence of the reciprocal of k2,app with [Na+]-2 was observed, indicating that at least two monovalent cation sites, or classes of sites, are necessary for the catalytic event to occur. From this latter plot, the k2,max for APC catalysis of NPGB hydrolysis, at saturating [Na+] and [NPGB], was calculated to be 1.21 +/- 0.10 s-1, and the Km for Na+ was found to be 21 +/- 3 mM. The dissociation constant, Ks, for NPGB to APC, at 7 degrees C, was not altered as [Na+] was increased, yielding a range of values of 18.5 X 10(-5) to 19.9 X 10(-5) M as [Na+] was varied from 12.5 to 62.5 mM. The deacylation rate constant, k3, for p-guanidinobenzoyl-APC hydrolysis was also independent of [Na+], with a value of (3.8 +/- 1.0) X 10(-3) s-1 in the absence of Na+ or in the presence of concentrations of Na+ up to 200 mM. Identical kinetic behavior was observed when Cs+ was substituted for Na+ in the above enzymic reaction. The pre-steady-state kinetic parameters were calculated according to the same methodology as described above.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distinction between the two basic mechanisms of cation transport in the cardiac Na(+)-Ca2+ exchange system

In order to distinguish between the Ping-Pong and sequential mechanisms of cation transport in the cardiac Na(+)-Ca2+ exchange system, the initial rates of the Nai-dependent 45Ca uptake (t = 1 s) were measured in reconstituted proteoliposomes, loaded with a Ca chelator. Under "zero-trans" conditions ([Na]o = [Ca]i = 0) at a fixed [Na]i = 10-160 mM with varying [45Ca]o = 2.5-122 microM for each [Na]i, the Km and Vmax values increased from 7.7 to 33.5 microM and from 2.3 to 9.0 nmol.mg-1.s-1, respectively. The Vmax/Km values show a +/- 2-10% deviation from the average value of 0.274 nmol.mg-1.s-1.microM-1 over the whole range of [Na]i. These deviations are within the standard error of Vmax (+/- 3-7%), Km (+/- 11-17%), and Vmax/Km (+/- 11-19%). This suggests that, under conditions in which Vmax and Km are [Na]i dependent and vary 4-5-fold, the Vmax/Km values are constant within the experimental error. In the presence of K(+)-valinomycin the Vmax/Km values are 0.85 +/- 0.17 and 1.08 +/- 0.18 nmol.mg-1.s-1.microM-1 at [Na]i = 20 and 160 mM, respectively, suggesting that under conditions of "short circuit" of the membrane potential the Vmax/Km values still exhibit the [Na]i independence. At a very low fixed [45Ca]o = 1.1 microM with varying [Na]i = 10-160 mM, the initial rates were found to be [Na]i independent. At a high fixed [45Ca]o = 92 microM the initial rates show a sigmoidal dependence on the [Na]i with Vmax = 13.8 nmol.mg-1.s-1, KmNa = 21 mM, and Hill coefficient nH = 1.5. The presented data support a Ping-Pong (consecutive) mechanism of cation transport in the Na(+)-Ca2+ exchanger.     

NMR studies of intracellular sodium ions in mammalian cardiac myocytes

The unambiguous measurement of intracellular sodium ion [Na+]i by the noninvasive NMR technique offers a new opportunity to monitor precisely the maintenance and fluctuations of [Na+]i levels in intact cells and tissues. The anionic frequency shift reagent, dysprosium (III) tripolyphosphate, which does not permeate intact cells, when added to suspensions of intact adult rat cardiac myocytes, alters the NMR frequency of extracellular sodium ions, [Na+]o, leaving that of intracellular ions, [Na+]i, unaffected. Using 23Na NMR in conjunction with this shift reagent, we have determined NMR-visible intracellular Na+ ion concentration in a suspension of isolated cardiac myocytes under standard conditions with insulin and Ca2+ in the extracellular medium to be 8.8 +/- 1.2 mmol/liter of cells (n = 4). This value is comparable to that measured by intracellular ion-selective microelectrodes in heart tissue. Cardiac myocytes incubated for several hours in insulin-deficient, Ca2+-containing medium prior to NMR measurement exhibited a somewhat lower [Na+]i value of 6.9 +/- 0.5 mmol/liter of cells (n = 3). Reversible Na+ loading of the cells by manipulation of extracellular calcium levels is readily measured by the NMR technique. Incubation of myocytes in a Ca2+-free, insulin-containing medium causes a 3-fold increase in [Na+]i to a level of 22.8 +/- 2.6 mmol/liter of cells (n = 10). In contrast to cells with insulin, insulin-deficient myocytes exhibit a markedly lower level of [Na+]i of only 14.6 +/- 2.0 mmol/liter of cells (n = 4) in Ca2+-free medium. These observations suggest that insulin may stimulate a pathway for Na+ influx in heart cells.     

Large changes in intracellular pH and calcium observed during heat shock are not responsible for the induction of heat shock proteins in Drosophila melanogaster.

Heat shock caused significant changes in intracellular pH (pHi) and intracellular free calcium concentration [( Ca2+]i) which occurred rapidly after temperature elevation. pHi fell from a resting level value at 25 degrees C of 7.38 +/- 0.02 (mean +/- standard error of the mean, n = 15) to 6.91 +/- 0.11 (n = 7) at 35 degrees C. The resting level value of [Ca2+]i in single Drosophila melanogaster larval salivary gland cells was 198 +/- 31 nM (n = 4). It increased approximately 10-fold, to 1,870 +/- 770 nM (n = 4), during a heat shock. When salivary glands were incubated in calcium-free, ethylene glycol-bis(beta-aminoethyl ether)-N,N',N'-tetraacetic acid (EGTA)-buffered medium, the resting level value of [Ca2+]i was reduced to 80 +/- 7 nM (n = 3), and heat shock resulted in a fourfold increase in [Ca2+]i to 353 +/- 90 nM (n = 3). The intracellular free-ion concentrations of Na+, K+, Cl-, and Mg2+ were 9.6 +/- 0.8, 101.9 +/- 1.7, 36 +/- 1.5, and 2.4 +/- 0.2 mM, respectively, and remained essentially unchanged during a heat shock. Procedures were devised to mimic or block the effects of heat shock on pHi and [Ca2+]i and to assess their role in the induction of heat shock proteins. We report here that the changes in [Ca2+]i and pHi which occur during heat shock are not sufficient, nor are they required, for a complete induction of the heat shock response.     

23Na NMR studies of rat outer medullary kidney tubules

Two reservations have previously made interpretation of biological 23Na NMR measurements difficult: the "size" of the extracellular space penetrated by the shift reagent and the possibility of a 60% reduction in the intensity of the NMR-visible 23Na signal due to quadrupolar interactions (Berendsen, H. J. C., and Edzes, H. T. (1973) Ann. N. Y. Acad. Sci. 204, 459-485; Civan, M. M., Degani, H., Margalit, Y., and Shporer, M. (1983) Am. J. Physiol. 245, C213-C219; Gupta, R. K., and Gupta, P. (1982) J. Magn. Reson. 47, 344-350). We have addressed both these issues using a suspension of rat outer medullary kidney tubules, nephron segments responsible for the fine control of total body volume and electrolyte balance. First, the extracellular space penetrated by the shift reagent dysprosium tripolyphosphate, as defined by the extracellular 23Na resonance, revealed a space similar to that which contained extracellular 35Cl- ions. Measurement of an extracellular 35Cl- space using 35Cl NMR was possible because the intracellular 35Cl- resonance was broadened beyond detection in the cells studied. Second, to characterize the reduction of the 23Na signal by quadrupolar interactions, the intracellular 23Na level was raised artificially by simultaneously inhibiting Na+ efflux and increasing the ion permeability of the plasma membrane. Under these conditions, NMR-observable intracellular Na+ reached a level which was approximately 81% of that in the medium, a level determined using chemical techniques. This observation would suggest that the resonance of the intracellular 23Na pool was not subject to a 60% reduction in signal intensity, as a result of nuclear quadrupolar interaction. The intracellular 23Na level measured, under basal conditions, was 23 +/- 2 mumol/ml of cell water (37 degrees C) (n = 3, S.D.) and was demonstrated to be responsive to a number of physiological stimuli. The level was temperature-sensitive. It was reduced by inhibitors of apical Na+ transport, furosemide and amiloride, and it was raised with (Na+ + K+)-ATPase inhibition. The furosemide and amiloride actions described would suggest that the Na+-transporting mechanisms sensitive to these agents (e.g. Na+/K+/Cl- cotransport system, Na+:H+ exchange system) contribute to the regulation of the intracellular Na+ level in the kidney tubular preparation studied.     

Multinuclear NMR studies of intracellular cations in perfused hypertensive rat kidney.

We have investigated hypertension-associated alterations in intracellular cations in the kidney by measuring intracellular pH, free Mg2+, free Ca2+, and Na+ concentrations in perfused normotensive and hypertensive rat (8-14 weeks old) kidneys using 31P, 19F, and double quantum-filtered (DQ) 23Na NMR. The effects of both anoxia and ischemia on the 23Na DQ signal confirmed its ability to detect changes in intracellular Na+. However, there was a sizable contribution of the extracellular Na+ to the 23Na DQ signal of the kidney. The intracellular free Ca2+ concentration, measured using 19F NMR and 5,5'difluoro-1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid, also increased dramatically during ischemia; the increase could be partly reversed by reperfusion. No significant differences were found between normotensive and hypertensive kidneys in the ATP level, intracellular pH, intracellular free Mg2+, and the 23Na DQ signal or in the extent of the extracellular contribution to the 23Na DQ signal. Oxygen consumption rates were also similar for the normotensive (5.02 +/- 0.46 mumol of O2/min/g) and hypertensive (5.47 +/- 0.42 mumol O2/min/g) rat kidneys. The absence of a significant difference in intracellular pH, Na+ concentration, and oxygen consumption between normotensive and hypertensive rat kidneys suggests that an alteration in the luminal Na+/H+ antiport activity in hypertension is unlikely. However, a highly significant increase (64%, p less than 0.01) in free Ca2+ concentration was found in perfused kidneys from hypertensive rats (557 +/- 48 nM, blood pressure = 199 +/- 5 mmHg, n = 6) compared with normotensive rats (339 +/- 21 nM, blood pressure = 134 +/- 6, n = 4) indicating altered renal calcium homeostasis in essential hypertension. An increase in intracellular free Ca2+ concentration without an accompanying change in the intracellular Na+ suggests, among many possibilities, that the Ca2+/Mg(2+)-ATPase may be inhibited in the hypertensive renal tissue.     

Double-quantum-filtered 23Na NMR study of intracellular sodium in the perfused liver.

We acquired double-quantum-filtered 23Na NMR spectra from perfused liver, using a range of tau values from 0.2 to 24 ms, where tau is the separation between the first and second pi/2 pulses in the radio-frequency pulse sequence. For each tau value we compared the amplitude of the double-quantum-filtered 23Na NMR signal acquired from intracellular sodium ions when the liver was perfused with buffer containing the "shift reagent" Dy(PPP)2 to the amplitude of the total double-quantum-filtered 23Na NMR signal acquired when the liver was perfused with buffer containing no Dy(PPP)2. For tau < or = 4 ms, the average ratio of the two amplitudes was 0.98 +/- 0.03 (mean +/- SEM). For tau > or = 8 ms, the average ratio was significantly less than 1. These results demonstrate that double-quantum-filtered 23Na NMR signals acquired from perfused liver using short tau values arise almost exclusively from intracellular sodium ions, but double-quantum-filtered 23Na NMR signals acquired from perfused liver using long tau values contain contributions from both intracellular and extracellular sodium ions. This conclusion suggests that multiple-quantum-filtered 23Na NMR spectroscopy will be useful in studying intracellular sodium levels in the perfused liver, and possibly in the intact liver in vivo.     

Use of 1,2-Bis(2-Amino-5-Fluorophenoxy)ethane-N,N,N'',N''-Tetraacetic Acid (5FBAPTA) in the Measurement of Free Intracellular Calcium in the Brain by 19F-Nuclear Magnetic Resonance Spectroscopy

We have applied the 19F-nuclear magnetic resonance (NMR) calcium indicator 1,2-bis(2-amino-5-fluoro-phenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA) to the measurement of the free intracellular calcium concentration [( Ca2+]i) in superfused brain slices. A mean +/- SD control value of 380 +/- 71 nM (n = 18) was obtained at 37 degrees C using 2.4 mM extracellular Ca2+. Subcellular fractionation studies using [3H]5FBAPTA showed that after loading of its tetraacetoxymethyl ester, approximately 55% was de-esterified, with the other 45% remaining as the tetraester bound to membranes. Of the de-esterified 5FBAPTA, greater than 90% was in the cytosolic fractions, with less than 1% in the mitochondria or microsomes. The NMR-visible de-esterified 5FBAPTA slowly disappeared from the tissue with a t1/2 of 4 h. A time course after loading confirmed that the calculated [Ca2+]i was constant over a 5-h period, although the scatter of individual results was +/- 20%. The [Ca2+]i was increased by a high extracellular K+ concentration ([K+]e), by a low extracellular concentration of Na+, and by the calcium ionophore A23187. On recovery from high [K+]e, the [Ca2+]i "overshot" to values lower than the original control value. The [Ca2+]i was surpisingly resistant to changes in extracellular Ca2+ concentration.     

Nuclear Magnetic Resonance Studies on the Effects of Decreased External Sodium on Guinea Pig Cerebral Cortex Slices and the Permeabilities of Various Sodium Substitutes

Decreasing the external sodium concentration ([Na+]e) to 10 mM in the presence of 280 mM sucrose had no significant effect on phosphocreatine (PCr) or on intracellular pH (pHi) as assessed using 31P nuclear magnetic resonance spectroscopy. Zero [Na+]e in the presence of 300 mM sucrose caused a fall in PCr levels to 50% of control values, and the pHi fell to 6.85 from a control value of 7.30. 1H nuclear magnetic resonance spectroscopy confirmed that the sucrose had not entered the tissue. The decreases in PCr content and in pHi, known to occur on depolarization using 40 mM external potassium concentration ([K+]e), were further decreased in the presence of 10 mM [Na+]e), to 51.4 +/- 4.0 and 6.80 +/- 0.10% of control values, respectively. The free intracellular magnesium concentration was significantly increased from a control value of 0.37 +/- 0.10 mM to 0.66 +/- 0.13 mM (p less than 0.001), when [Na+]e was decreased to 10 mM, but was not further affected by high [K+]e or zero Na+. Membrane permeabilities of the sodium substitutes N-methyl-D-glucamine (NMG), tris(hydroxymethyl)aminomethane (Tris), tetramethylammonium (TMA), and choline were assessed using 1H nuclear magnetic resonance spectroscopy. In the presence of 10 mM [Na+]e, NMG, TMA, and choline (all at 140 mM) were taken up and remained within the tissue for at least 2 h, but no uptake of Tris (140 mM) or sucrose (above) could be detected. Tissue lactate levels (from the lactate/N-acetyl aspartate ratio) increased in the presence of the substitutes that were taken up, although no change in pH was detected.     

Intracellular sodium affects ouabain interaction with the Na/K pump in cultured chick cardiac myocytes

Whether a given dose of ouabain will produce inotropic or toxic effects depends on factors that affect the apparent affinity (K0.5) of the Na/K pump for ouabain. To accurately resolve these factors, especially the effect of intracellular Na concentration (Nai), we have applied three complementary techniques for measuring the K0.5 for ouabain in cultured embryonic chick cardiac myocytes. Under control conditions with 5.4 mM Ko, the value of the K0.5 for ouabain was 20.6 +/- 1.2, 12.3 +/- 1.7, and 6.6 +/- 0.4 microM, measured by voltage-clamp, Na-selective microelectrode, and equilibrium [3H]ouabain-binding techniques, respectively. A significant difference in the three techniques was the time of exposure to ouabain (30 s-30 min). Since increased duration of exposure to ouabain would increase Nai, monensin was used to raise Nai to investigate what effect Nai might have on the apparent affinity of block by ouabain. Monensin enhanced the rise in Na content induced by 1 microM ouabain. In the presence of 1 microM [3H]ouabain, total binding was found to be a saturating function of Na content. Using the voltage-clamp method, we found that the value of the K0.5 for ouabain was lowered by nearly an order of magnitude in the presence of 3 microM monensin to 2.4 +/- 0.2 microM and the magnitude of the Na/K pump current was increased about threefold. Modeling the Na/K pump as a cyclic sequence of states with a single state having high affinity for ouabain shows that changes in Nai alone are sufficient to cause a 10-fold change in K0.5. These results suggest that Nai reduces the value of the apparent affinity of the Na/K pump for ouabain in 5.4 mM Ko by increasing its turnover rate, thus increasing the availability of the conformation of the Na/K pump that binds ouabain with high affinity.     

Nuclear magnetic resonance of 23Na ions interacting with the gramicidin channel.

Basic nuclear magnetic resonance (NMR) features of 23Na ions bound to the gramicidin channel (packaged into lecithin liposomes) were studied. The first binding constant K1 of Na+ was not significantly dependent on channel models employed. With the two-identical-site model (Model I), K1 was 13.7 (+/- 1.4) molal-1 (in the activity basis) at 25 degrees C; when the binding of a third ion was included (Model II), it was 13.0 (+/- 2.0) molal-1. The second binding constant K2 was model dependent; it was 1.6 (+/- 0.2) and 3-4 molal-1 for Models I and II, respectively. The rate constants, k-1 and k-2, of Na+ for exit from singly and doubly loaded channels, respectively, were 8 X 10(5) s-1 less than or equal to k-1 less than or equal to 3 X 10(6) s-1 and 8 X 10(5) s-1 less than or equal to k-2 less than or equal to 1.0 X 10(7) s-1 at 25 degrees C; the lower bound represents a rough approximation of k-1. The ratio k-2/k-1 was greater than one and did not greatly exceed 20. From the competition experiment, K1 of T1+ was 5.7 (+/- 0.6) X 10(2) molal-1. The longitudinal relaxation time T1 of bound 23Na in the state of single occupancy (T 1B sing) was virtually independent of models, 0.56 (+/- 0.03) and 0.55 (+/- 0.04) ms at 25 degrees C for Models I and II, respectively. For the state of double occupancy, T1 of bound 23Na (T 1B doub) was model dependent: 0.27 (+/- 0.01) and 0.4-0.6 ms for Models I and II. The correlation time tau c of bound 23Na was 2.2 (+/- 0.2) ns at 25 degrees C for single occupancy; tau c for double occupancy was not significantly different from this value. The estimated tau c was found to involve no appreciable contribution of the exchange of 23Na between the channel and the bulk solution. Thé quadrupole coupling constant chi was 1.0 (+/- 0.1) MHz for 23Na in single occupancy; chi for double occupancy was 0.9-1.4 MHz, depending on models. A lower bound of the average quadrupole coupling constant chi alpha was 0.13-0.26 MHz at 25 degrees C for 23Na in single occupancy; this value represents a rough approximation of chi alpha at this temperature. An argument based on the estimated chi alpha and the known conformation of the gramicidin channel suggests that the binding site is a small domain near the channel end.(ABSTRACT TRUNCATED AT 400 WORDS)     

Multiple coherence of cerebral blood flow velocity in humans

The coherence function has been used in transfer function analysis of dynamic cerebral autoregulation to assess the statistical significance of spectral estimates of gain and phase frequency response. Interpretation of the coherence function and choice of confidence limits has not taken into account the intrinsic nonlinearity represented by changes in cerebrovascular resistance due to vasomotor activity. For small spontaneous changes in arterial blood pressure (ABP), the relationship between ABP and cerebral blood flow velocity (CBFV) can be linearized, showing that corresponding changes in cerebrovascular resistance should be included as a second input variable. In this case, the standard univariate coherence function needs to be replaced by the multiple coherence, which takes into account the contribution of both inputs to explain CBFV variability. With the use of two different indicators of cerebrovascular resistance index [CVRI = ABP/CBFV and the resistance-area product (RAP)], multiple coherences were calculated for 42 healthy control subjects, aged 20 to 40 yr (28 +/- 4.6 yr, mean +/- SD), at rest in the supine position. CBFV was measured in both middle cerebral arteries, and ABP was recorded noninvasively by finger photoplethysmography. Results for the ABP + RAP inputs show that the multiple coherence of CBFV for frequencies <0.05 Hz is significantly higher than the corresponding values obtained for univariate coherence (P < 10(-5)). Corresponding results for the ABP + CVRI inputs confirm the principle of multiple coherence but are less useful due to the interdependence between CVRI, ABP, and CBFV. The main conclusion is that values of univariate coherence between ABP and CBFV should not be used to reject spectral estimates of gain and phase, derived from small fluctuations in ABP, because the true explained power of CBFV in healthy subjects is much higher than what has been usually predicted by the univariate coherence functions.     

Intracellular pH-regulating mechanism of the squid axon. Interaction between DNDS and extracellular Na+ and HCO3-

Intracellular pH (pHi) of the squid axon is regulated by a stilbenesensitive transporter that couples the influx of Na+ and HCO3- (or the equivalent) to the efflux of Cl-. According to one model, the extracellular ion pair NaCO3- exchanges for intracellular Cl-. In the present study, the ion-pair model was tested by examining the interaction of the reversible stilbene derivative 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) with extracellular Na+ and HCO3-. Axons (initial pHi approximately 7.4) were internally dialyzed with a pH 6.5 solution containing 400 mM Cl- but no Na+. After pHi, as measured with a glass microelectrode, had fallen to approximately 6.6, dialysis was halted. In the presence of both external Na+ and HCO3- (pHo = 8.0, 22 degrees C), pHi increased due to the pHi-regulating mechanism. At a fixed [Na+]o of 425 mM and [HCO3-]o of 12 mM, DNDS reversibly reduced the equivalent acid-extrusion rate (JH) calculated from the rate of pHi recovery. The best-fit value for maximal inhibition was 104%, and for the [DNDS]o at half-maximal inhibition, 0.3 mM. At a [Na+]o of 425 mM, the [HCO3-]o dependence of JH was examined at 0, 0.1, and 0.25 mM DNDS. Although Jmax was always approximately 20 pmol cm-2 s-1, Km(HCO3-) was 2.6, 5.7, and 12.7 mM, respectively. Thus, DNDS is competitive with HCO3-. At a [HCO3-]o of 12 mM, the [Na+]o dependence of JH was examined at 0 and 0.1 mM DNDS. Although Jmax was approximately 20 pmol cm-2 s-1 in both cases, Km(Na+) was 71 and 179 mM, respectively. At a [HCO3-]o of 48 mM, Jmax was approximately 20 pmol cm-2 s-1 at [DNDS]o levels of 0, 0.1, and 0.25 mM. However, Km(Na+) was 22, 45, and 90 mM, respectively. Thus, DNDS (an anion) is also competitive with Na+. The results are consistent with simple competition between DNDS and NaCO3-, and place severe restrictions on other kinetic models.     

Calcium influx in internally dialyzed squid giant axons

A method has been developed to measure Ca influx in internally dialyzed squid axons. This was achieved by controlling the dialyzed segment of the axon exposed to the external radioactive medium. The capacity of EGTA to buffer all the Ca entering the fiber was explored by changing the free EGTA at constant [Ca++]i. At a free [EGTA]i greater than 200 microM, the measured resting Ca influx and the expected increment in Ca entry during electrical stimulation were independent of the axoplasmic free [EGTA]. To avoid Ca uptake by the mitochondrial system, cyanide, oligomycin, and FCCP were included in the perfusate. Axons dialyzed with a standard medium containing: [ATP] = 2 mM, [Ca++]i = 0.06 microM, [Ca++]o = 10 mM, [Na+]i = 70 mM, and [Na+]o = 465 mM, gave a mean Ca influx of 0.14 +/- 0.012 pmol.cm-2.s-1 (n = 12. Removal of ATP drops the Ca influx to 0.085 +/- 0.007 pmol.cm-2.s-1 (n = 12). Ca influx increased to 0.35 pmol.cm-2,s-1 when Nao was removed. The increment was completely abolished by removing Nai+ and (or) ATP from the dialysis medium. At nominal zero [Ca++]i, no Nai-dependent Ca influx was observed. In the presence of ATP and Nai [Ca++]i activates the Ca influx along a sigmoid curve without saturation up to 1 microM [Ca++]i. Removal of Nai+ always reduced the Ca influx to a value similar to that observed in the absence of [Ca++]i (0.087 +/- 0.008 pmol.cm-2.s-1; n = 11). Under the above standard conditions, 50-60% of the total Ca influx was found to be insensitive to Nai+, Cai++, and ATP, sensitive to membrane potential, and partially inhibited by external Co++.     

23Na and 39K nuclear magnetic resonance studies of perfused rat hearts. Discrimination of intra- and extracellular ions using a shift reagent.

High-resolution 23Na and 39K nuclear magnetic resonance (NMR) spectra of perfused, beating rat hearts have been obtained in the absence and presence of the downfield shift reagent Dy(TTHA)3- in the perfusing medium. Evidence indicates that Dy(TTHA)3- enters essentially all extracellular spaces but does not enter intracellular spaces. It can thus be used to discriminate the resonances of the ions in these spaces. Experiments supporting this conclusion include interventions that inhibit the Na+/K+ pump such as the inclusion of ouabain in and the exclusion of K+ from the perfusing medium. In each of these experiments, a peak corresponding to intracellular sodium increased in intensity. In the latter experiment, the increase was reversed when the concentration of K+ in the perfusing medium was returned to normal. When the concentration of Ca2+ in the perfusing medium was also returned to normal, the previously quiescent heart resumed beating. In the beating heart where the Na+/K+ pump was not inhibited, the intensity of the intracellular Na+ resonance was less than 20% of that expected. Although the data are more sparse, the NMR visibility of the intracellular K+ signal appears to be no more than 20%.