Effects of temperature on ionic transport aortas from rat and ground squirrel KRISTINE E. KAMM, MARVIN L. ZATZMAN, ALLAN AND FRANK E. SOUTH Department of Physiology, University of Missouri Medical Center, University of Missouri, Columbia, Missouri 65212
KAMM,KRISTINE E., MARVIN L. ZATZMAN, ALLAN W. JONES, AND FRANK E. SOUTH. Effects of temperature on ionic transport in aortas from rat and ground squirrel. Am. J. Physiol. 237(l): C23-C30, 1979 or Am. J. Physiol.: Cell Physiol. 6( 1): C23-C30, 1979.-Isotopic efflux of sodium and potassium were measured in aortic smooth muscle from the rat and the ground squirrel, a hibernator, at temperatures between 37OC and OOC. Turnover of 42K in cells of hibernators was significantly lower at 0°C than in the cells of rats. Qlo values of 1.5 at temperatures above 10°C for the hibernator and the rat suggest potassium moved passively out of cells. 24Na turnover in K-free solution was highly temperature dependent in all groups; Q10 between 2.6 and 3.7 imply that this passive movement of sodium was not by free diffusion. K-stimulated 24Na turnover at 8OC was significantly greater in the aortas from both normothermic and hibernating squirrels than in those from rats. Turnover followed Arrhenius’ theory between 37OC and 8OC in squirrel tissues. However, in the rat vessel the Arrhenius plot demonstrated a distinct break at 17OC, at which temperature the Q10 increased more than threefold to 8.8. It is suggested that cells of the hibernator may have undergone alterations in lipids which maintain “fluidity” in the domain of the transport sites during cold. A model is proposed to explain the maintenance of ionic gradients at 7°C by the hibernator. An important factor is the stimulation of active transport by a small increase in cell
IW= active Na’ turnover
transport;
smooth
muscle;
hibernator;
potassium
CORTEX and red blood cells (20, 26), a major physiological characteristic distinguishing aortic smooth muscle of hibernators from that of nonhibernators is the ability of the former to maintain ion concentration gradients in the cold. In a previous study (18) it was observed that aortic cells from rats lost potassium (K) and gained sodium (Na) with a half time of 11-14 h during 7OC incubation in oxygenated Krebs solution. Under the same conditions, cells from ground squirrel aortas retained these ions. It has been suggested (27) that inhibition of cellular functions in the cold is the result of a primary effect on energy expending activities of the cell rather than on those supplying energy. In this light, it is proposed that control of ionic gradients in the smooth muscle of a hibernator at low temperature is due to specialized adAS IN KIDNEY
in
W. JONES,
Center and the Dalton
Research
aptation of sites controlling ionic transport. Isotope movements of ions were measured at temperatures between 37 and O°C in rat and ground squirrel aortas. The ability of tissues from a hibernator to maintain concentration gradients in the cold could result from an adaptation of the cellular membrane in two ways: I) decrease in membrane permeability with cooling, and/or 2) maintanence of the Na-K pump at low temperature. The present work indicates that a combination of both mechanisms is involved. METHODS
Animals
and Tissue Preparation
Three groups of animals were used in this study. Male Wistar rats were chosen as the nonhibernating species. Hibernators, 13.lined ground squirrel (CiteZZus tridecemZineatus) of mixed sex were divided into normothermic and hibernating groups. The hibernating group was maintained at 4OC and were hibernating at least 48 h before being killed. This group had been through at least two cycles of hibernation. Tissues were prepared as described previously (18). Briefly, the aortas were removed and cleaned of loose connective tissue in a dissection solution at 37OC. Tissues from hibernating squirrels were dissected at l°C. Strips were cut along the long axis, mounted on stainless steel holders, and incubated in Krebs solution for 3 h.
Incubation
Media
All solutions were prepared with double distilled water and analytical grade reagents. The Krebs solution had the following composition (mM): Na’ 146.2, K+ 5.0, Mg2+ 1.2, Ca2+ 2.5, Cl- 143.9, HCOT 13.5, H2POQ 1.2, and glucose 5.6. Solutions were gassed with 9’7% 02-3s Co2 maintaining a pH of 7.4. The K+ and Ca”’ were replaced with Na’ in the dissection solution. This reversibly depleted the aortas of endogenous K+ which allowed the tissues to come into isotopic equilibrium with 42K during the subsequent 3-h incubation. K-free Krebs solution had Na substituted for K. The loading solutions for washout experiments contained lo-20 ,&i/ml of either 24Na or 42K. Isotopes were obtained from the University of Missouri Research Reactor.
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Radioisotope
KAMM,
Experiments
Isotope washout techniques were similar to those described previously (13, 17). Strips from normothermic squirrels and rats were placed in 30 ml of loading solution at 37*C for 3 h. Vessels from hibernating animals were incubated in loading solutions at 4*C for 5 h. The tissues were then rinsed l-2 s in nonlabeled Krebs solution to remove surface fluid, and passed through a series of tubes, each containing 5 ml nonradioactive solution. Tissue weight rarely exceeded 25 mg, thus maintaining a medium to tissue ra tio of 2OO:l The solution within the tubes was stirred by vigorously bubbling with the O&O2 gas. The temperature of washout solutions was controlled by immersing tubes in thermostatically regulated water baths. The activity in the tubes and tissues was counted in a gamma well counter for 42K. At the end of an experiment, the tissue was removed from the holder, blotted on filter paper and weighed. Tissue electrolytes were extracted into 5 ml of 0.1 N HN03, 10 mM La3’, and 15 mM Li+ in NO3 form for counting and subsequent ionic analysis. Tissues and solutions containing 24Na were counted using liquid scintillation techniques. The electrolytes were extracted as for 42K samples. After 30 min the extract plus tissue were transferred to a counting vial, neutralized with 0.5 ml of 1 N NaOH, and mixed with 10 ml of scintillation fluid (Research Products International, RPI 3A70B). Quenching was uniform in all the samples as measured by a channel ratio method. Washout curves were derived by sequentially adding the counts (corrected for decay) in the tissue and tubes. The fraction of the counts remaining during the washout was calculated by dividing the count after each period by the initial count. The rate constant, k (min-‘), for isotope loss during each period was also computed. 42K washout. Tissues were washed for 30 min at several temperatures (37”, 31*, 25*, 15”, 7*, 5”, and O*C). Protocols began with 37°C and sequentially passed the tissue through lower temperatures. Finally, the tissues were returned to 37*C to compare rate constants at the beginning and end of the experiment. They routinely agreed within 10%. Rate constants were derived from a leastsquares fit to an exponential function during the last three periods at each temperature. 24Na washout. Aortas were equilibrated 3 h in 24Na Krebs solution at 37”C, then kept at a lower temperature for an additional 10 min. The tissues were washed out at O*C or 4*C for 40 min, followed by a higher temperature (31°C or 37OC) for 20 min. All strips were then reincubated in normal 24Na Krebs solution, at 37”C, for 1 h. This allowed the ionic and isotopic steady state to be reestablished. The tissues were then washed out at a difTerent low (8°C or 12*C) and high (17°C or 25°C) temperature. Thus, 24Na turnover was determined at four temperatures for each tissue. Half of each aorta was washed out in normal Krebs solution and the other half in K-free solution. Tissue counts for the first washout were estim .ated from the count i .n the next to last tube since pilot studi .es demonstrated no difference (P > 0.5, after 60 min of n = 8) between them. Furthermore, washout at O*C or 4*C and 31°C or 37°C the tissue count
ZATZMAN,
JONES,
AND
SOUTH
was only 0.1-0.5s of the initial count. Therefore this estimate contributed less than 1% variation to the reconstruction of the washout curve. Rate constants were calculated by linear regression techniques applied to the fraction of count remaining at 20, 30, and 40 min for low temperatures and 40, 42, and 45 min for high temperatures. The rate associated with active Na transport at a given temperature was calculated as the difference in rate constants between the halfaortas washed into K-free solution and those washed into Krebs solution containing 5 mM K. Data were plotted according to the Arrhenius equation for a reaction following the relation: k = Ae-“(l? A plot of the log of k versus l/T (OK) yields a straight line with the slope -E,/R, E, being the activation energy of the reaction and R is the gas constant. Values for E, were calculated from fitted slopes according to the equation
E a = R(ln kTz - ln kT,)/(TI’ In addition
the Qlo was calculated
Q10 Statistical
- ?fz’)
as
10
kT,
= (T 2-
TI)
“T,
Analysis
Analysis of variance tables were constructed using a Statistical Analysis Systems program. Log-linear models were used to test for difference in Arrhenius slopes between groups. The level of significance was chosen as P < 0.05. Control samples were compared to experimental samples by means of the Student’s t test. RESULTS
42K Washout A slower passive efflux of K at low temperature could underlie the ability of the squirrel aorta to maintain high K in comparison to the rat. This was tested by measuring the effects of temperature on the washout of ‘*K. Serial reduction in temperature (Fig. 1A) reduced the 42K washout from rat aorta except at the lowest temperature. Washouts conducted at one temperature (Fig. 1B) exhibited essentially single exponential behavior with the rate constants being similar to those derived at the same temperature during serial reduction in temperature. The changes in rate constants observed in Fig. 1A therefore, do not arise from some time-dependent nonlinearity in the 42K washout but reflect direct effects of temperature. Arrhenius plots of the rate constants appear in Fig. 2A for the rat and Fig. 2B for the ground squirrel. Under the experimental conditions, the maximal decrease in cell K is about 10% (11, 18) making this a negligible factor compared to the effects of temperature on the rate constants, thus, changes in the rate constant reflect changes in K efflux. As temperature was reduced, the rate constants for rat aortas fell from a peak of 0.0094 t 0.0005 min-’ (n = 12) at 37*C to a nadir of 0.0022 t 0.0004 min-* (n = 9) at 7*C. Rate constants decreased exponentially (r = 0.93) between 37°C and 15°C. Below 7°C the rate constants tended to rise, reaching 0.0032 t 0.0004 min-’
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A
c25
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100 r
FIG. 1. A: washout of 42K from representative rat aorta at four temperatures. Counts are expressed as a percent of initial counts (logarithmic scale) and plotted vs. time. Arrows indicate time of temperature change. B: washout of 42K fromrataoti~at370C(*)(n=5)and
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(0) (n = 5) plotted as in of initial counts (logarithmic time. Vertical bars are f: SE.
7OC
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?
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*
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20
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9
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200
37 min
250
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B
Rat Aorta
Wormothermic Proud Squirrel Aorta
-2
-25-
FIG. 2. A: Arrhenius plot of 42K turnover (log k vs. l/T) from aortas of rats. Each point is the mean and vertical bars are SE. Number of animals are 12, 6, 6, 5, 4, 6, and 9 at 37, 31, 25, 15, 7, 4, and O”C, respectively. The Qlo between 37 and 15°C is 1.4. B: Arrhenius plot of 42K turnover (log k vs. l/T) from aortas of normothermic ground squirrels. Each point is the mean and vertical bars are SE. Number of animals are 15,6,9,6,8, 6, and 12 at 37, 31, 25, 15, 7, 4, and OOC, respectively. The 910 between 37 and 10°C is 1.4.
s-
= 3 C Y ,” W 0
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30 I 33
I 34
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1. Energy of activation and Q10 (n = 9) at OOC. The &lo and apparent EQ of this, and the TABLE following experiments, are summarized in Table 1. The for aortic ion turnover rate of 42K washout from normothermic ground squirrels Regression Coef, Apparent E,,, (Fig. 2B) decreased exponentially between 37OC and 7OC kcal/mol (r) (r = 0.92). The rate constant was 0.0092 t 0.0004 min-’ Experimental (n = 10) at 37OC and reached a minimum of 0.0016 t procedure 0.0002 min-’ (n = 8) at 7OC. Values below 7OC were not 42K Turnover significantly different. The rate constants from hibernat0.93 10 Rat ing ground squirrels were the same as the normothermic Normothermic 0.92 10 group* ground squirrel The linear portions of the Arrhenius plots from both Hibernating ground 0.85 10 squirrel hibernating and normothermic squirrels were not different from the rat (P > 0.8), but the rate constant of the K-free 24Na turnover rat vessel was significantly higher than that of both 0.98 18 Rat Normothermic 0.96 15 groups of squirrels at O°C (P < O.Ol), perhaps indicating ground squirrel some difference in permeability at this temperature.
“Na
vs.
t
0
-
scale)
zI-
.-c E
a =
A: percent
Wmhout
Decreased inhibition of active Na transport at low temperature could also underlie the ability of the squirrel aorta to maintain low Na (and high K) in comparison to the rat. This was tested by measuring the effects of temperature on the washout of 24Na. A representative washout from a rat aorta is shown in Fig. 3. At low temperatures a slowly exchanging component (l-2%) could be identified. The rate was similar in the presence
Hibernating squirrel
K-Stimulated turnover Rat
ground
Temp Range, “C
1.4 1.4
37-15 37-7
1.5
37-7
3.7 2.7
37-4 37-4
0.95
15
2.6
37-4
0.79 (0.90) * 0.92 0.93
9 (14)* 36 15
1.4 (2.4)* 8.8 2.5
37-17 17-4 37-4
0.85
12
2.0
37-8
24Na
Normothermic ground squirrel Hibernating ground squirrel ( )* Based on corrected value; see DIscussIoN).
rate
constant
at 37°C
(1.75
x
measured
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KAMM,
and absence of K. When the tissues were warmed to 37°C the rates increased, but in the presence of K the rate was about twice that in K-free solution. It is assumed that this difference was the result of active transport. 24Na washout in K-free solutions. 24Na turnover under K-free conditions primarily represents passive movement of Na from the cells, due mainly to Na:Na exchange, with a smaller leak and perhaps, a Na:Ca exchange component (1). The Arrhenius plots of “4Na turnover in aortas from rats and ground squirrels appear in Fig. 4. At 37*C the rate constants were 0.143 t 0.121 min-’ (n = 7) for the rat, and 0.110 t 0.12 (n = 6) and 0.095 t 0.008 min-’ (n = 5) for normothermic and hibernating ground squirrels, respectively. The rate constants decreased in a monotonic fashion with decreasing temperature. No distinct break was evident. The rate constants at 4*C were 0.0042 t 0.0006 min-’ (n = 6) for the rat, and 0.0055 t 0.0010 (n = 6) and 0.0055 t 0.0004 min-’ (n = 7) for normothermic and hibernating ground squirrels. No significant differences between the three groups were noted. The E, and Qlo for 24Na turnover in K-free solutions were 1.5-2 times greater than the corresponding values for 42K turnover (Table 1) in the three groups (P < 0.01). K-stimulated 24Na washout. The Arrhenius plot of rate constants for K-stimulated Na transport in rat aortas exhibited a distinct break at 17°C (Fig. 5). Rate constants decreased from 0.081 t 0.012 min-’ (n = 7) at 37*C to 0.031 2 0.006 min-’ (n = 7) at 4*C. The Qlo ranged
A0
30
ZATZMAN,
20
JONES,
10
AND
SOUTH
0
tw
FIG. 4. Arrhenius plot of “Na turnover in K-free Krebs. Each point is the mean and vertical bars are SE. Measurements were made at 37, 31, 26, 17, 12,8, and 4°C. Number of animals at each point: Rats (0) 7, 6, 6, 5, 6, 6, and 6 (corrected value (x), see Discussion); normothermic ground squirrels; (A) and thin dashed line, 6, 6, 5, 5, 5, 7, and 6; hibernating squirrels, (0) and dashed line, 5, 8, 6, 6, 6, 6, and 7.
40
60
Minutes 3. 24Na washout Erom a rat aorta into 0 mM or 5 mM K Krebs at 4 and 37°C. Counts are expressed as a percent of initial counts (logarithmic scale) and plotted against time. Arrow indicates time of transfer to 37°C. K-stimulated 24Na turnover was calculated as the difference in rate between the half aorta washed in K-free Krebs and that washed in 5 mM K Krebs. Note that the slow component (the intercept of the regression line through 20, 30, and 40 min) is l-2% of the initial counts. FIG.
between 1.4 and 2.4 (Table 1). At 17*C the Arrhenius slope increased abruptly; the rate constant dropped to a minimum of 0.0024 t 0.0008 min-’ (n = 7) at 4*C. The &lo (Table 1) b e t ween 17°C and 4*C increased over threefold compared to that between 37*C and 17°C (P < 0.001). Freeman-Narrod and Goodford (8) observed a similar phenomenon in guinea pig tenia coli where there was little difference between 42K uptake at 35°C and 2O”C, but this process was greatly slowed at 4°C. Jones (11) also observed relatively small effects on “4Na turnover over the 37-20°C range in rabbit myometrium compared to uptake at low temperatures. In contrast to the rat, the Arrhenius plot for normothermic squirrel aortas was linear (r = 0.93) over the 37-4°C range as shown in Fig. 6. Rate constants decreased from 0.108 t 0.009 min (n = 6) at 37°C to 0.0063 t 0.0009 min-’ (n = 7) at 4*C. Rate constants of K-stimulated Na transport in hibernating squirrel aortas fell linearly over the 37-8°C range (r = 0.85). There was, however, an abrupt fall at 4*C to 0.0025 t 0.0006 min-’ (n = 7). The &lo and apparent E, for the three preparations were similar over the 37.17°C range (Table 1). There is some uncertainty in the rat because of the change in [Nalcell (See Discussion). Both groups of squirrels had a much lower &lo than rats over the 17.8*C temperature range (P < 0.001). At 8”C, the transport rate constant
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C27 (P < 0.02) after an additional 6 h at 7OC in 24Na. No further increase was observed after 48 h at 7OC. DISCUSSION
42K Washout T F Rat Aorta
*< 4
\ I \
\ I
4.0 c 32
3.0
?O
I 3.3
I 3.4
-
0 (ocJ .
‘P I 3.5
1 3.6
The aim of this investigation was to examine mechanisms which contribute to the maintenance of ionic gradients in aortic smooth muscle of ground squirrels incubated in the cold. The hypothesis that cells of hibernators were less permeant to potassium at low temperature than rats was tested by comparing 42K turnover. The rate constant for 42K turnover by rat aorta at O°C was significantly greater than that for both groups of squirrels; however there was no significant difference between values of rat and squirrel at 7OC and 4°C. Kimzey and Willis (21) concluded that reduced passive permeability at low temperatures is one mechanism whereby the hibernator maintains the ionic composition of red blood cells. The trend here appears to be the same, but it is difficult to make firm conclusions about the significance of the contribution of such an adaptation to the maintenance of ionic gradients in the hibernator’s vascular muscle. The &lo was similar between 37°C and 10°C in all groups (Table 1). The low Qlo (1.4-1.5) is similar to that of a diffusional process and similar values for 42K rate con-
1 3.7
I x 103 IoK-‘1 1
FIG. 5. Arrhenius plot of K-stimulated 24Na turnover in rat aortas. Each point (A) is the mean and vertical bars are SE (corrected value (x), see Discussion). Number of animals are 7, 6, 5, 4, 6, 6, and 7 at 37, 31,26,17,12,8, and 4”C, respectively. Note the change in slope at 17°C.
for aortas from normothermic squirrels, 0.0095 t 0.0016 b-’ (n = 6), was not significantly different from that for the hibernating squirrels, 0.0128 t 0.0025 min-’ (n = 5) (P > 0.2). However, both were significantly greater than that for the rat, 0.0049 t 0.0011 min-’ (n = 6) (P < 0.05). Slowly exchanging sodium. It was possible to find the initial count of slowly exchanging 24Na at 37OC by extrapolation of the washout to zero time (Fig. 3). The sodium concentration in cell water, [Na],,u, was derived from the specific activity of the isotope solution and the fraction of tissue weight comprising cell water: 0.21 for rats and 0.25 for ground squirrels (18). The [Nalceu was not different between rat and normothermic squirrels (Table 2). Aortas, that were taken from hibernating squirrels and maintained at 4”C, exhibited a significant increase in [Na]cell. It was of interest to determine the effects of acute cold on [Na]cell. This was done on the rat aorta by comparing the slowly exchanging 24Na after 3 h at 37OC and 10 min at l”C, to that after an additional 40 min at l°C in 24Na. Increased time in the cold was associated with a 75% increase in [Na]cell from 14 t 2 (n = 9) to 25 t 4 (n = 9) mmol/l cell Hz0 (P < 0.025). In the normothermic ground squirrel, [Na]cell increased from 9.0 AZ0.5 (n = 6) at 37°C to 12.1 t 0.8 mmol/l cell Hz0
f
40 I 32
\
IJormothermit Ground Squirrel Aorta 4
30
20
I 3.3
I 3.3
T
1
10 I 3.5
0 I 3.6
i"cl 1 3.7
)( 103 (OK-‘1
FIG. 6. Arrhenius plot of K-stimulated ‘L4Na turnover in aortas from normothermic ground squirrels. Each point is the mean and vertical bars are SE. Number of animals are 6, 6, 4, 4, 4, 6, and 7 at 37, 31, 26, 17,12,8, and 4”C, respectively.
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KAMM,
TABLE
2. %w~y
exchanging
Group
Ftat aorta Normothermic ground . squirrel aorta Hibernating ground squirrel aorta
cell sodium Slow [ Na), ,.I)
mmol/I cell Hz0
12.2 k 1.0 12.7 t, 2.0
(11)
23.5 A 3.5
u3)*
(9)
(P < 0.05)
&ants were found in tenia coli (2). At temperatures below lO*C, rate constants increased in the rat cells while a plateau was approached by the squirrel aorta. Daniel (5) speculated that the unexpectedly high rate of potassium loss from rat uterus between 19°C and 6*C resulted in part from membrane depolarization occurring during sudden cooling. In the present experiments, tissues were transferred from 37*C through intermediate temperatures before being exposed to 7”C, 4”C, or O*C. It seems unlikely that the high rates observed at very low temperatures resulted from depolarization stimulated by sudden cooling. Cells may have been depolarized by inhibition of sodium pumping at low temperature; however the transition temperature of Na transport occurred at 17°C while K turnover continued to fall between 17*C and 10°C. Although depolarization is a possible factor, it is unlikely that the increased K turnover at low temperatures was an indirect response to the effect of cold on the membrane potential. Temperature studies on nonirritable tissues demonstrated a similar phenomenon; passive K influx into dog red cells increased below 17°C (7). 24Na Washout The second set of experiments was designed to study the effects of temperature on both active and passive transport of 24Na. The K-insensitive washout of 24Na was 64% of the totaI in the rat tissue at 37*C and 51% in the squirrel tissue. Half of the Na efflux from Na-rich tenia coli was concluded to be ouabain insensitive Na-exchange diffusion (4). The 24Na turnover in K-free solution followed Arrhenius theory; the decline was linear from 37 to 4°C for all three groups. None showed a distinct break or transition temperature. Brading (1) also observed the K-insensitive 24Na turnover to be highly temperature dependent in tenia coli. The Q1o for the aortic tissues from the rat and squirrels were far from the value suggested for free diffusion. This implies that this exchange involved membrane-molecular interactions. It was postulated that K-stimulated sodium transport of squirrel cells would be less inhibited by cold than that of rat cells. At 8*C Na transport from both normothermic and hibernating squirrels was significantly greater than the rat. A distinct break occurred in the Arrhenius plot at 17°C for the rat aorta. A threefold increase in Qlo for active transport occurred below this temperature. Kstimulated sodium transport in squirrel vessels, however, followed Arrhenius theory over the 37.8*C temperature range. Because of the linear decline in rate, at lower temperatures the turnover exceeded that in the rat. This had adaptive significance for maintenance of ionic gradients in the cold. Furthermore, this adaptation was present in normothermic squirrels, which indicates it was
ZATZMAN,
JONES,
AND
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not acquired by recent bouts of hypothermia. Kimzey and Willis (21) demonstrated that ouabain-sensitive sodium efflux from guinea pig erythrocytes was affected more by a decrease of temperature than was the sodium efflux from ground squirrel erythrocytes. This adaptation appears to be shared by excitable and nonexcitable tissues in the hibernator. In the present studies, K-stimulated Na transport fell linearly with temperature in the normothermic squirrel tissue, but fell off rapidly below 8*C in the hibernating squirrel. The implication of this observation is not clear. It seems unlikely that the membrane became “deadapted.” It is noted, however, that the low rate constant occurs below the temperature the animals attain when hibernating (20), and therefore may not affect the ability of the vessel to perform ionic transport during hibernation. Analysis of the effects of temperature on active transport were complicated by two problems. First, the rate of active Na efflux can only be estimated as a difference between total and inhibited unidirectional fluxes. Because of the insensitivity of the rat to ouabain (6) the Kdependent flux was used as an estimate of this function. This requires the [K]o surrounding the cells to be negligible during the K-free washout. This can be checked by estimating the maximum accumulation of K in the extracellular space. On return to 37°C the loss of K is about 1% per minute for tenia coli and myometrium (3, 12), which is equivalent to 1.5 mM fall in cell K during the first minute. Extracellular volume is twice intracellular (18); therefore in the case of no exchange, [K]o would increase by only 0.75 mM. However, diffusional delays in rat aorta of 140 ,um thickness (16) are small, having a half time of less than 10 s for 42K at 37*C (14). With more than 6 half times/min the [K]o at 37°C would be less than 10% of that above. According to the shape of the activation curve (15), [K]o would have to be in the 0.5 mM range to stimulate active transport. We assume then, that under the present conditions [K]o is not sufficiently high in K-free solutions to stimulate Na extrusion significantly. A second problem is that the tissues are no longer in a steady state when cooled (18). This is especially important for the rat. If during 40 min of washout at low temperature [Nalcell rises, then the rate constant at 37*C may not be an accurate reflection of the sodium efflux. The additional 40 min at 1°C was associated with a 75% increase in [Na],,u. The efflux at 37*C (rate x [Na]& wou ld therefore be gre tater than inferred fro m the plot of rate constants alone. Both measured and a corrected (1.75 x measured) value are included in Figs. 4 and 5 to give the maximum range. For low [NalCell, the rate should be rel .atively constant for small changes in [Nalcell, yielding a proportionality between [Na] cell and efflux (9, 15, 22). The measured value is probably the more reliable one. Although this factor limits detailed comparisons over the upper temperature range, the more critical comparisons at low temperatures are relatively unaffected because they were derived shortly after loading - in 24Na at 37*C. Aside from showing qu antitative differences in ionic tu rno,ver 1between groups, Arrhenius plots provide some
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GROUND
SQUIRREL
insight into the mechanism(s) by which aortic smooth muscle from the hibernator is adapted to support function at low temperature. Departures from linearity in Arrhenius plots have been interpreted in a number of ways. The effect of temperature on K-stimulated Na transport resulted in a monotonic decline in ionic turnover in the hibernator, while a break or increased E, occurred at 17°C in the nonhibernator. This may suggest that the phospholipids associated with the transport enzyme in the hibernator may be adapted to maintain fluidity at low temperatures. This postulate is supported by other findings of modifications in tissue lipid composition in hibernating mammals (10, 19, 24, 25). Implications can also be drawn from the differences between the effects of temperature on 42K turnover, Kfree 24Na turnover, and K-stimulated 24Na turnover. The Arrhenius plots for the rat vessels each have a unique configuration. One might conclude that the unidirectional fluxes occur at different sites in the membrane. Second, because the transition temperatures for 42K turnover (10°C) and K-stimulated 24Na turnover (17OC) are different, it is speculated that if these breaks in Arrhenius plots are due to phase changes in lipids, the phase change occurs only in the domain of the transport site. The domain concept is further supported by the observation that the 42K turnover is not in agreement with Arrhenius theory between 10°C and 0°C; turnover actually increases over this range. Lyons (23) postulated that a change in state could bring about a contraction of the membrane, causing cracks or channels to appear, which could lead to an increase in permeability. However, this seems unlikely since the loss of sodium does not increase at these temperatures. Finally, 24Na turnover in a K-free medium decreased linearly from 37OC to 4OC, in accord with Arrhenius theory. It seems probable that K-insensitive Na exchange occurs by ion exchange mechanisms separate from the Na-K pump. This is in agreement with the conclusions of Brading (1).
Flux Imbalance
c29
AORTA
at Low Temperature
An effort was made to synthesize the information on fluxes into a model to predict changes in ionic gradients at low temperature (Fig. 7). Measured K-stimulated Na efflux and rate constants for 42K washout were used to predict ionic fluxes on an hourly basis. The rate constants measured on tissues suddenly cooled to 7OC were assumed to be constant during prolonged cold. At least two factors may affect this assumption. One was the unknown effect of long-term cooling on membrane permeability, and the other was the increase in [Na],,n. Jones (15 and unpublished observations) has found the K-stimulated sodium efflux from the rat aorta to be proportional to [Nalcen over the lower range (12-25 mM). Saturation was approached above 25 mM. With cell sodium at low concentrations it was reasonable to assume that the rate constant remained invariant. The ratio of K efflux (assumed to be equal to K influx in steady state) and Kstimulated Na efflux at 37OC was unity in both the rat and squirrel tissues. It was assumed that this was also the case at 7OC and that the ratio remained unchanged during cold incubation. Finally, it was assumed that
Ground
Squirrel
Aorta
3Wa 1~1 coupling
Time
CKI cell
h-1
INaGe
1
3K pump
Hr-')
(.594
(fl/l/hr) 0
K lost Ic Na aained
a (.096
(fl)
Active Na efflux
K efflux
147
73
14x
140.6
19.4
13.49
Hr-') (rPl/l/hr)
7.72
6.4
11.52
2.0
2
138.6
21.4
13.31
12.71
.6
3
138.0
22.0
13.24
13.07
.2
4
137.8
22.2
13.23
13.19
.04
Theoretical
steady
state_
7Oc 4 hours
FIG. 7. Flux imbalance at 7OC in the ground squirrel aorta. Effluxes were calculated from the product of rate constants (h-‘) and intracellular ionic concentrations (Table 1, Ref. 18). The difference between active Na efflux (assumed equivalent to K influx) and K efflux was assumed equal to potassium lost per hour. This amount was subtracted from original cell K and added to cell Na. The subsequent fluxes were reiteratively calculated from the new ionic concentrations. When active Na efflux was equal to K efflux the cells were in a new steady state.
energy for transport was not limiting. Squirrel cells were in a “pump-leak” imbalance upon initial exposure to the cold. For approximately 5 K leaking out only 3 K were pumped in. Figure 7 predicts that as ionic concentrations change, the imbalance will decrease until a new steady state is achieved at 138 mM K and 22 mM Na, involving a shift of 10 mM Na and K during cold incubation (18). A direct check of the predictions were made by measuring slowly exchanging 24Na at 37 and 7OC. The increase in [Nalcell was complete by 6 h and stable up to 48 h at 7°C. Although the change in [Nalcell was only about one third that calculated in Fig. 7, the evidence supports the conclusion that the most important factor in bringing the tissues into a steady state at low temperature is the stimulation of active transport by elevated [Nalcell. Rat cells were also in a pump-leak imbalance on exposure to cold. For every 8 K leaking only 1 K was replaced. If all assumptions were correct, the rat tissue should have approached a new steady state of 105 mM K and 59 mM Na after 8 h in the cold. These levels were not in accord with observed values after 48 h of cold incubation (18). The discrepancy is attributable in part to saturation of the active transport mechanism before a pump-leak balance can be achieved. Other contributions
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (128.059.222.107) on January 15, 2019.
c30
KAMM,
to the inability of these cells to retain ionic gradients might be a changing K permeability in the cold and/or inadequate metabolic support for transport. Additional experiments are needed before a quantitative description of pump-leak imbalance can be made for the rat. We thank Ms. Jeannette petent assistance and Dr. squirrels used in this study.
Kilfoil and Shu X. J. Musacchia
Hsien Liu Tsay for comfor donating the ground
ZATZMAN,
JONES,
AND
SOUTH
This work was supported by Public Health Service Grants HL17847, HL-07094, and HL-15852, and by the Research Council of the Graduate-School of the University of Missouri. A. W. Jones acknowledges the American Heart Association for his Established Investigator Fellowship. Present addresses: K. E. Kamm, Laboratorium voor Fysiologie, Campus Gasthuisberg, K.U.L., B-3000 Leuven, Belgium, and F. E. South, School of Life and Health Sciences, University of Delaware, Newark, DE 19717. Received
26 June
1978; accepted
in final
form
22 January
1979.
REFERENCES
BRADING, A. F. Sodium/sodium exchange in the smooth muscle of the guinea pig taenia coli. J. Physiol. London 251: 79-105, 1975. 2. BRADING, A., E. BULBRING, AND T. TOMITA. The effect of temper-
1.
3.
4.
5.
6.
7.
8.
9.
10.
11.
ature on the membrane conductance of the smooth muscle of the guinea pig, taenia coli. J. Physiol. London 200: 621-635, 1969. CASTEELS, R., G. DROOGMANS, AND H. HENDRICKX. Membrane potential of smooth muscle cells in K-free solution. J. Physiol. London 217: 281-295, 1971. CASTEELS, R., G. DROOGMANS, AND H. HENDRICKX. Effect of sodium and sodium-substitutes on the active ion transport and on the membrane potential of smooth muscle cells. J. Physiol. London 228: 733-748, 1973. DANIEL, E. E. Potassium movements in rat uterus studied in vitro. I. Effects of temperature. Can. J. Biochem. Physiol. 41: 2065-2084, 1963. DANIEL, E. E. The interconnection between active transport and contractures in uterine tissues. Can. J. Biochem. Physiol. 42: 453495, 1964. ELFORD, B. C. Interactions between temperature and tonicity on cation transport in dog red cells. J. Physiol. London 246: 371-395, 1975. FREEMAN-NARROD, M., AND P. J. GOODFORD. Sodium and potassium content of the smooth muscle of the guinea-pig taenia coli at different temperatures and tensions. J. Physiol. London 163: 399410, 1962. GARRAHAN, P. J., AND R. P. GARAY. A kinetic study of the Na pump in red cells: its relevance to the mechanism of active transport. Ann. NY Acad. Sci. 242: 445-458, 1974. GOLDMAN, S. S. Cold resistance of the brain during hibernation. III. Evidence of a lipid adaptation. Am. J. Physiol. 228: 834-838,
1975. JONES, A. W. Factors
affecting sodium exchange and distribution myometrium. Physiol. Chem. Phys. 2: 79-95, 1970. 12. JONES, A. W. Effects of progesterone treatment on potassium accumulation and permeation in rabbit myometrium. Physiol. Chem. Phys. 2: 151-167, 1970. 13. JONES, A. W. Altered ion transport in vascular smooth muscle from spontaneously hypertensive rats: influence of aldosterone, norepinephrine and angiotensin. Circ. Res. 33: 563-572, 1973. 14. JONES, A. W. Analysis of bulk diffusion limited exchange of ions. In: Methods in Pharmacology. Smooth Muscle, edited by E. E. Daniel and D. M. Patton. New York: Plenum, 1975, chapt. 39, p. 673-687. in rabbit
15. JONES, A. W. K-stimulated Na transport in aortic smooth muscle and changes with DOCA hypertension in rats. Federation Proc. 35: 698, 1976. 16. JONES, A. W., P. D. SANDER, AND D. L. KAMPSCHMIDT. The effect of norepinephrine on aortic 42K turnover during deoxycorticosterone acetate hypertension and antihypertensive therapy in the rat. Circ. Res. 41: 256-260, 1977. 17. JONES, A. W., AND M. L. SWAIN. Chemical and kinetic analyses of sodium distribution in canine lingual artery. Am. J. Physiol. 223: 1110-1118, 1972. 18. KAMM, K. E., M. L. ZATAMAN, A. W. JONES, AND F. E. SOUTH. Maintenance of ion concentration gradients in the cold in aorta from rat and ground squirrel. Am. J. Physiol. 237: C17-C22, 1979 or Am. J. Physiol.: Cell Physiol. 6: Cl 7-C22, 1979. 19. KEITH, A. D., R. C. ALOIA, J. LYONS, W. SNIPES, AND E. T. PENGELLEY. Spin label evidence for the role of lysoglycerophosphatides in cellular membranes of hibernating mammals. Biochim. Biophys. Acta 394: 204-210, 1975. 20. KIMZEY, S. L., AND J. S. WILLIS. Resistance of erythrocytes of hibernating mammals to loss of potassium during hibernation and during cold storage. J. Gen. Physiol. 58: 620-633, 1971. 21. KIMZEY, S. L., AND J. S. WILLIS. Temperature adaptation of active sodium-potassium transport and of passive permeability in eryth’rocytes of ground squirrels. J. Gen. Physiol. 58: 634-649, 1971. 22. KNIGHT, A. B., AND L. G. WELT. Intracellular potassium: a determinant of the sodium-potassium pump rate. J. Gen. Physiol. 63: 351-373, 1974. 23. LYONS, J. Phase transitions and control of cellular metabolism at low temperatures. Cryobiology 9: 341-350, 1972. 24. MCMURCHIE, E. J., AND J. K. RAISON. Hibernation and homeothermic status of the Echidna (Tachyglossus aculeatus). J. Therm. BioZ. 1: 113-118, 1976. 25. RAISON, J. K., AND 3. M. LYONS. Hibernation: alteration of mitochondrial membranes as a requisite for metabolism at low temperature. Proc. Nat. Acad. Sci. USA 68: 2092-2094, 1971. 26. WILLIS, J. S. Characteristics of ion transport in kidney cortex of mammalian hibernators. J. Gen. Physiol. 49: 1221-1239, 1966. 27. WILLIS, J. S., L. S. T. FANG, AND R. F. FOSTER. The significance and analysis of membrane function in hibernation. In: Hibernation and Hypothermia, edited by F. E. South, J. P. Hannon, J. R. Willis, E. T. Pengelley, and N. R. Alpert. Amsterdam: Elsevier, 1972, p. 123-147.
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KAMM,KRISTINE E., MARVIN L. ZATZMAN, ALLAN W. JONES, AND FRANK E. SOUTH. Effects of temperature on ionic transport in aortas from rat and ground squirrel. Am. J. Physiol. 237(l): C23-C30, 1979 or Am. J. Physiol.: Cell Physiol. 6( 1): C23-C30, 1979.-Isotopic efflux of sodium and potassium were measured in aortic smooth muscle from the rat and the ground squirrel, a hibernator, at temperatures between 37OC and OOC. Turnover of 42K in cells of hibernators was significantly lower at 0°C than in the cells of rats. Qlo values of 1.5 at temperatures above 10°C for the hibernator and the rat suggest potassium moved passively out of cells. 24Na turnover in K-free solution was highly temperature dependent in all groups; Q10 between 2.6 and 3.7 imply that this passive movement of sodium was not by free diffusion. K-stimulated 24Na turnover at 8OC was significantly greater in the aortas from both normothermic and hibernating squirrels than in those from rats. Turnover followed Arrhenius’ theory between 37OC and 8OC in squirrel tissues. However, in the rat vessel the Arrhenius plot demonstrated a distinct break at 17OC, at which temperature the Q10 increased more than threefold to 8.8. It is suggested that cells of the hibernator may have undergone alterations in lipids which maintain “fluidity” in the domain of the transport sites during cold. A model is proposed to explain the maintenance of ionic gradients at 7°C by the hibernator. An important factor is the stimulation of active transport by a small increase in cell
IW= active Na’ turnover
transport;
smooth
muscle;
hibernator;
potassium
CORTEX and red blood cells (20, 26), a major physiological characteristic distinguishing aortic smooth muscle of hibernators from that of nonhibernators is the ability of the former to maintain ion concentration gradients in the cold. In a previous study (18) it was observed that aortic cells from rats lost potassium (K) and gained sodium (Na) with a half time of 11-14 h during 7OC incubation in oxygenated Krebs solution. Under the same conditions, cells from ground squirrel aortas retained these ions. It has been suggested (27) that inhibition of cellular functions in the cold is the result of a primary effect on energy expending activities of the cell rather than on those supplying energy. In this light, it is proposed that control of ionic gradients in the smooth muscle of a hibernator at low temperature is due to specialized adAS IN KIDNEY
in
W. JONES,
Center and the Dalton
Research
aptation of sites controlling ionic transport. Isotope movements of ions were measured at temperatures between 37 and O°C in rat and ground squirrel aortas. The ability of tissues from a hibernator to maintain concentration gradients in the cold could result from an adaptation of the cellular membrane in two ways: I) decrease in membrane permeability with cooling, and/or 2) maintanence of the Na-K pump at low temperature. The present work indicates that a combination of both mechanisms is involved. METHODS
Animals
and Tissue Preparation
Three groups of animals were used in this study. Male Wistar rats were chosen as the nonhibernating species. Hibernators, 13.lined ground squirrel (CiteZZus tridecemZineatus) of mixed sex were divided into normothermic and hibernating groups. The hibernating group was maintained at 4OC and were hibernating at least 48 h before being killed. This group had been through at least two cycles of hibernation. Tissues were prepared as described previously (18). Briefly, the aortas were removed and cleaned of loose connective tissue in a dissection solution at 37OC. Tissues from hibernating squirrels were dissected at l°C. Strips were cut along the long axis, mounted on stainless steel holders, and incubated in Krebs solution for 3 h.
Incubation
Media
All solutions were prepared with double distilled water and analytical grade reagents. The Krebs solution had the following composition (mM): Na’ 146.2, K+ 5.0, Mg2+ 1.2, Ca2+ 2.5, Cl- 143.9, HCOT 13.5, H2POQ 1.2, and glucose 5.6. Solutions were gassed with 9’7% 02-3s Co2 maintaining a pH of 7.4. The K+ and Ca”’ were replaced with Na’ in the dissection solution. This reversibly depleted the aortas of endogenous K+ which allowed the tissues to come into isotopic equilibrium with 42K during the subsequent 3-h incubation. K-free Krebs solution had Na substituted for K. The loading solutions for washout experiments contained lo-20 ,&i/ml of either 24Na or 42K. Isotopes were obtained from the University of Missouri Research Reactor.
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C24
Radioisotope
KAMM,
Experiments
Isotope washout techniques were similar to those described previously (13, 17). Strips from normothermic squirrels and rats were placed in 30 ml of loading solution at 37*C for 3 h. Vessels from hibernating animals were incubated in loading solutions at 4*C for 5 h. The tissues were then rinsed l-2 s in nonlabeled Krebs solution to remove surface fluid, and passed through a series of tubes, each containing 5 ml nonradioactive solution. Tissue weight rarely exceeded 25 mg, thus maintaining a medium to tissue ra tio of 2OO:l The solution within the tubes was stirred by vigorously bubbling with the O&O2 gas. The temperature of washout solutions was controlled by immersing tubes in thermostatically regulated water baths. The activity in the tubes and tissues was counted in a gamma well counter for 42K. At the end of an experiment, the tissue was removed from the holder, blotted on filter paper and weighed. Tissue electrolytes were extracted into 5 ml of 0.1 N HN03, 10 mM La3’, and 15 mM Li+ in NO3 form for counting and subsequent ionic analysis. Tissues and solutions containing 24Na were counted using liquid scintillation techniques. The electrolytes were extracted as for 42K samples. After 30 min the extract plus tissue were transferred to a counting vial, neutralized with 0.5 ml of 1 N NaOH, and mixed with 10 ml of scintillation fluid (Research Products International, RPI 3A70B). Quenching was uniform in all the samples as measured by a channel ratio method. Washout curves were derived by sequentially adding the counts (corrected for decay) in the tissue and tubes. The fraction of the counts remaining during the washout was calculated by dividing the count after each period by the initial count. The rate constant, k (min-‘), for isotope loss during each period was also computed. 42K washout. Tissues were washed for 30 min at several temperatures (37”, 31*, 25*, 15”, 7*, 5”, and O*C). Protocols began with 37°C and sequentially passed the tissue through lower temperatures. Finally, the tissues were returned to 37*C to compare rate constants at the beginning and end of the experiment. They routinely agreed within 10%. Rate constants were derived from a leastsquares fit to an exponential function during the last three periods at each temperature. 24Na washout. Aortas were equilibrated 3 h in 24Na Krebs solution at 37”C, then kept at a lower temperature for an additional 10 min. The tissues were washed out at O*C or 4*C for 40 min, followed by a higher temperature (31°C or 37OC) for 20 min. All strips were then reincubated in normal 24Na Krebs solution, at 37”C, for 1 h. This allowed the ionic and isotopic steady state to be reestablished. The tissues were then washed out at a difTerent low (8°C or 12*C) and high (17°C or 25°C) temperature. Thus, 24Na turnover was determined at four temperatures for each tissue. Half of each aorta was washed out in normal Krebs solution and the other half in K-free solution. Tissue counts for the first washout were estim .ated from the count i .n the next to last tube since pilot studi .es demonstrated no difference (P > 0.5, after 60 min of n = 8) between them. Furthermore, washout at O*C or 4*C and 31°C or 37°C the tissue count
ZATZMAN,
JONES,
AND
SOUTH
was only 0.1-0.5s of the initial count. Therefore this estimate contributed less than 1% variation to the reconstruction of the washout curve. Rate constants were calculated by linear regression techniques applied to the fraction of count remaining at 20, 30, and 40 min for low temperatures and 40, 42, and 45 min for high temperatures. The rate associated with active Na transport at a given temperature was calculated as the difference in rate constants between the halfaortas washed into K-free solution and those washed into Krebs solution containing 5 mM K. Data were plotted according to the Arrhenius equation for a reaction following the relation: k = Ae-“(l? A plot of the log of k versus l/T (OK) yields a straight line with the slope -E,/R, E, being the activation energy of the reaction and R is the gas constant. Values for E, were calculated from fitted slopes according to the equation
E a = R(ln kTz - ln kT,)/(TI’ In addition
the Qlo was calculated
Q10 Statistical
- ?fz’)
as
10
kT,
= (T 2-
TI)
“T,
Analysis
Analysis of variance tables were constructed using a Statistical Analysis Systems program. Log-linear models were used to test for difference in Arrhenius slopes between groups. The level of significance was chosen as P < 0.05. Control samples were compared to experimental samples by means of the Student’s t test. RESULTS
42K Washout A slower passive efflux of K at low temperature could underlie the ability of the squirrel aorta to maintain high K in comparison to the rat. This was tested by measuring the effects of temperature on the washout of ‘*K. Serial reduction in temperature (Fig. 1A) reduced the 42K washout from rat aorta except at the lowest temperature. Washouts conducted at one temperature (Fig. 1B) exhibited essentially single exponential behavior with the rate constants being similar to those derived at the same temperature during serial reduction in temperature. The changes in rate constants observed in Fig. 1A therefore, do not arise from some time-dependent nonlinearity in the 42K washout but reflect direct effects of temperature. Arrhenius plots of the rate constants appear in Fig. 2A for the rat and Fig. 2B for the ground squirrel. Under the experimental conditions, the maximal decrease in cell K is about 10% (11, 18) making this a negligible factor compared to the effects of temperature on the rate constants, thus, changes in the rate constant reflect changes in K efflux. As temperature was reduced, the rate constants for rat aortas fell from a peak of 0.0094 t 0.0005 min-’ (n = 12) at 37*C to a nadir of 0.0022 t 0.0004 min-* (n = 9) at 7*C. Rate constants decreased exponentially (r = 0.93) between 37°C and 15°C. Below 7°C the rate constants tended to rise, reaching 0.0032 t 0.0004 min-’
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ION
TRANSPORT
IN
RAT
AND
GROUND
SQUIRREL
A
c25
AORTA
B
100 r
FIG. 1. A: washout of 42K from representative rat aorta at four temperatures. Counts are expressed as a percent of initial counts (logarithmic scale) and plotted vs. time. Arrows indicate time of temperature change. B: washout of 42K fromrataoti~at370C(*)(n=5)and
*\0 37t
50 -
-\
"i \
0
25.c ?%
t if
* 0 p i i ii i i 1 f i i 1 1 1 P [ ?
20 1 -\ 37Oc 4 *\
i
i
(0) (n = 5) plotted as in of initial counts (logarithmic time. Vertical bars are f: SE.
7OC
4
I
?
10 -
*
*
0
I
I
I
20
A
-201
\
1
60
!
I
I
l4Omln
100
I
1
I
50
0
1
100
I
150
7”c
9
’
200
37 min
250
-2o-
0 \
B
Rat Aorta
Wormothermic Proud Squirrel Aorta
-2
-25-
FIG. 2. A: Arrhenius plot of 42K turnover (log k vs. l/T) from aortas of rats. Each point is the mean and vertical bars are SE. Number of animals are 12, 6, 6, 5, 4, 6, and 9 at 37, 31, 25, 15, 7, 4, and O”C, respectively. The Qlo between 37 and 15°C is 1.4. B: Arrhenius plot of 42K turnover (log k vs. l/T) from aortas of normothermic ground squirrels. Each point is the mean and vertical bars are SE. Number of animals are 15,6,9,6,8, 6, and 12 at 37, 31, 25, 15, 7, 4, and OOC, respectively. The 910 between 37 and 10°C is 1.4.
s-
= 3 C Y ,” W 0
-30
J
-3 40 1
I 32
30 I 33
I 34
1
P
10
20
T
1 35
x lo3 (OK-',
I 36
PC 1 37
1
o40
I 32
30
20
I 33
I 34
I 35
1 T
x
10
lo3
P
I 36
PC)
1 37
(OK-‘1
1. Energy of activation and Q10 (n = 9) at OOC. The &lo and apparent EQ of this, and the TABLE following experiments, are summarized in Table 1. The for aortic ion turnover rate of 42K washout from normothermic ground squirrels Regression Coef, Apparent E,,, (Fig. 2B) decreased exponentially between 37OC and 7OC kcal/mol (r) (r = 0.92). The rate constant was 0.0092 t 0.0004 min-’ Experimental (n = 10) at 37OC and reached a minimum of 0.0016 t procedure 0.0002 min-’ (n = 8) at 7OC. Values below 7OC were not 42K Turnover significantly different. The rate constants from hibernat0.93 10 Rat ing ground squirrels were the same as the normothermic Normothermic 0.92 10 group* ground squirrel The linear portions of the Arrhenius plots from both Hibernating ground 0.85 10 squirrel hibernating and normothermic squirrels were not different from the rat (P > 0.8), but the rate constant of the K-free 24Na turnover rat vessel was significantly higher than that of both 0.98 18 Rat Normothermic 0.96 15 groups of squirrels at O°C (P < O.Ol), perhaps indicating ground squirrel some difference in permeability at this temperature.
“Na
vs.
t
0
-
scale)
zI-
.-c E
a =
A: percent
Wmhout
Decreased inhibition of active Na transport at low temperature could also underlie the ability of the squirrel aorta to maintain low Na (and high K) in comparison to the rat. This was tested by measuring the effects of temperature on the washout of 24Na. A representative washout from a rat aorta is shown in Fig. 3. At low temperatures a slowly exchanging component (l-2%) could be identified. The rate was similar in the presence
Hibernating squirrel
K-Stimulated turnover Rat
ground
Temp Range, “C
1.4 1.4
37-15 37-7
1.5
37-7
3.7 2.7
37-4 37-4
0.95
15
2.6
37-4
0.79 (0.90) * 0.92 0.93
9 (14)* 36 15
1.4 (2.4)* 8.8 2.5
37-17 17-4 37-4
0.85
12
2.0
37-8
24Na
Normothermic ground squirrel Hibernating ground squirrel ( )* Based on corrected value; see DIscussIoN).
rate
constant
at 37°C
(1.75
x
measured
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (128.059.222.107) on January 15, 2019.
C26
KAMM,
and absence of K. When the tissues were warmed to 37°C the rates increased, but in the presence of K the rate was about twice that in K-free solution. It is assumed that this difference was the result of active transport. 24Na washout in K-free solutions. 24Na turnover under K-free conditions primarily represents passive movement of Na from the cells, due mainly to Na:Na exchange, with a smaller leak and perhaps, a Na:Ca exchange component (1). The Arrhenius plots of “4Na turnover in aortas from rats and ground squirrels appear in Fig. 4. At 37*C the rate constants were 0.143 t 0.121 min-’ (n = 7) for the rat, and 0.110 t 0.12 (n = 6) and 0.095 t 0.008 min-’ (n = 5) for normothermic and hibernating ground squirrels, respectively. The rate constants decreased in a monotonic fashion with decreasing temperature. No distinct break was evident. The rate constants at 4*C were 0.0042 t 0.0006 min-’ (n = 6) for the rat, and 0.0055 t 0.0010 (n = 6) and 0.0055 t 0.0004 min-’ (n = 7) for normothermic and hibernating ground squirrels. No significant differences between the three groups were noted. The E, and Qlo for 24Na turnover in K-free solutions were 1.5-2 times greater than the corresponding values for 42K turnover (Table 1) in the three groups (P < 0.01). K-stimulated 24Na washout. The Arrhenius plot of rate constants for K-stimulated Na transport in rat aortas exhibited a distinct break at 17°C (Fig. 5). Rate constants decreased from 0.081 t 0.012 min-’ (n = 7) at 37*C to 0.031 2 0.006 min-’ (n = 7) at 4*C. The Qlo ranged
A0
30
ZATZMAN,
20
JONES,
10
AND
SOUTH
0
tw
FIG. 4. Arrhenius plot of “Na turnover in K-free Krebs. Each point is the mean and vertical bars are SE. Measurements were made at 37, 31, 26, 17, 12,8, and 4°C. Number of animals at each point: Rats (0) 7, 6, 6, 5, 6, 6, and 6 (corrected value (x), see Discussion); normothermic ground squirrels; (A) and thin dashed line, 6, 6, 5, 5, 5, 7, and 6; hibernating squirrels, (0) and dashed line, 5, 8, 6, 6, 6, 6, and 7.
40
60
Minutes 3. 24Na washout Erom a rat aorta into 0 mM or 5 mM K Krebs at 4 and 37°C. Counts are expressed as a percent of initial counts (logarithmic scale) and plotted against time. Arrow indicates time of transfer to 37°C. K-stimulated 24Na turnover was calculated as the difference in rate between the half aorta washed in K-free Krebs and that washed in 5 mM K Krebs. Note that the slow component (the intercept of the regression line through 20, 30, and 40 min) is l-2% of the initial counts. FIG.
between 1.4 and 2.4 (Table 1). At 17*C the Arrhenius slope increased abruptly; the rate constant dropped to a minimum of 0.0024 t 0.0008 min-’ (n = 7) at 4*C. The &lo (Table 1) b e t ween 17°C and 4*C increased over threefold compared to that between 37*C and 17°C (P < 0.001). Freeman-Narrod and Goodford (8) observed a similar phenomenon in guinea pig tenia coli where there was little difference between 42K uptake at 35°C and 2O”C, but this process was greatly slowed at 4°C. Jones (11) also observed relatively small effects on “4Na turnover over the 37-20°C range in rabbit myometrium compared to uptake at low temperatures. In contrast to the rat, the Arrhenius plot for normothermic squirrel aortas was linear (r = 0.93) over the 37-4°C range as shown in Fig. 6. Rate constants decreased from 0.108 t 0.009 min (n = 6) at 37°C to 0.0063 t 0.0009 min-’ (n = 7) at 4*C. Rate constants of K-stimulated Na transport in hibernating squirrel aortas fell linearly over the 37-8°C range (r = 0.85). There was, however, an abrupt fall at 4*C to 0.0025 t 0.0006 min-’ (n = 7). The &lo and apparent E, for the three preparations were similar over the 37.17°C range (Table 1). There is some uncertainty in the rat because of the change in [Nalcell (See Discussion). Both groups of squirrels had a much lower &lo than rats over the 17.8*C temperature range (P < 0.001). At 8”C, the transport rate constant
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (128.059.222.107) on January 15, 2019.
ION
TRANSPORT
IN
RAT
AND
GROUND
SQUIRREL
AORTA
C27 (P < 0.02) after an additional 6 h at 7OC in 24Na. No further increase was observed after 48 h at 7OC. DISCUSSION
42K Washout T F Rat Aorta
*< 4
\ I \
\ I
4.0 c 32
3.0
?O
I 3.3
I 3.4
-
0 (ocJ .
‘P I 3.5
1 3.6
The aim of this investigation was to examine mechanisms which contribute to the maintenance of ionic gradients in aortic smooth muscle of ground squirrels incubated in the cold. The hypothesis that cells of hibernators were less permeant to potassium at low temperature than rats was tested by comparing 42K turnover. The rate constant for 42K turnover by rat aorta at O°C was significantly greater than that for both groups of squirrels; however there was no significant difference between values of rat and squirrel at 7OC and 4°C. Kimzey and Willis (21) concluded that reduced passive permeability at low temperatures is one mechanism whereby the hibernator maintains the ionic composition of red blood cells. The trend here appears to be the same, but it is difficult to make firm conclusions about the significance of the contribution of such an adaptation to the maintenance of ionic gradients in the hibernator’s vascular muscle. The &lo was similar between 37°C and 10°C in all groups (Table 1). The low Qlo (1.4-1.5) is similar to that of a diffusional process and similar values for 42K rate con-
1 3.7
I x 103 IoK-‘1 1
FIG. 5. Arrhenius plot of K-stimulated 24Na turnover in rat aortas. Each point (A) is the mean and vertical bars are SE (corrected value (x), see Discussion). Number of animals are 7, 6, 5, 4, 6, 6, and 7 at 37, 31,26,17,12,8, and 4”C, respectively. Note the change in slope at 17°C.
for aortas from normothermic squirrels, 0.0095 t 0.0016 b-’ (n = 6), was not significantly different from that for the hibernating squirrels, 0.0128 t 0.0025 min-’ (n = 5) (P > 0.2). However, both were significantly greater than that for the rat, 0.0049 t 0.0011 min-’ (n = 6) (P < 0.05). Slowly exchanging sodium. It was possible to find the initial count of slowly exchanging 24Na at 37OC by extrapolation of the washout to zero time (Fig. 3). The sodium concentration in cell water, [Na],,u, was derived from the specific activity of the isotope solution and the fraction of tissue weight comprising cell water: 0.21 for rats and 0.25 for ground squirrels (18). The [Nalceu was not different between rat and normothermic squirrels (Table 2). Aortas, that were taken from hibernating squirrels and maintained at 4”C, exhibited a significant increase in [Na]cell. It was of interest to determine the effects of acute cold on [Na]cell. This was done on the rat aorta by comparing the slowly exchanging 24Na after 3 h at 37OC and 10 min at l”C, to that after an additional 40 min at l°C in 24Na. Increased time in the cold was associated with a 75% increase in [Na]cell from 14 t 2 (n = 9) to 25 t 4 (n = 9) mmol/l cell Hz0 (P < 0.025). In the normothermic ground squirrel, [Na]cell increased from 9.0 AZ0.5 (n = 6) at 37°C to 12.1 t 0.8 mmol/l cell Hz0
f
40 I 32
\
IJormothermit Ground Squirrel Aorta 4
30
20
I 3.3
I 3.3
T
1
10 I 3.5
0 I 3.6
i"cl 1 3.7
)( 103 (OK-‘1
FIG. 6. Arrhenius plot of K-stimulated ‘L4Na turnover in aortas from normothermic ground squirrels. Each point is the mean and vertical bars are SE. Number of animals are 6, 6, 4, 4, 4, 6, and 7 at 37, 31, 26, 17,12,8, and 4”C, respectively.
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (128.059.222.107) on January 15, 2019.
C28
KAMM,
TABLE
2. %w~y
exchanging
Group
Ftat aorta Normothermic ground . squirrel aorta Hibernating ground squirrel aorta
cell sodium Slow [ Na), ,.I)
mmol/I cell Hz0
12.2 k 1.0 12.7 t, 2.0
(11)
23.5 A 3.5
u3)*
(9)
(P < 0.05)
&ants were found in tenia coli (2). At temperatures below lO*C, rate constants increased in the rat cells while a plateau was approached by the squirrel aorta. Daniel (5) speculated that the unexpectedly high rate of potassium loss from rat uterus between 19°C and 6*C resulted in part from membrane depolarization occurring during sudden cooling. In the present experiments, tissues were transferred from 37*C through intermediate temperatures before being exposed to 7”C, 4”C, or O*C. It seems unlikely that the high rates observed at very low temperatures resulted from depolarization stimulated by sudden cooling. Cells may have been depolarized by inhibition of sodium pumping at low temperature; however the transition temperature of Na transport occurred at 17°C while K turnover continued to fall between 17*C and 10°C. Although depolarization is a possible factor, it is unlikely that the increased K turnover at low temperatures was an indirect response to the effect of cold on the membrane potential. Temperature studies on nonirritable tissues demonstrated a similar phenomenon; passive K influx into dog red cells increased below 17°C (7). 24Na Washout The second set of experiments was designed to study the effects of temperature on both active and passive transport of 24Na. The K-insensitive washout of 24Na was 64% of the totaI in the rat tissue at 37*C and 51% in the squirrel tissue. Half of the Na efflux from Na-rich tenia coli was concluded to be ouabain insensitive Na-exchange diffusion (4). The 24Na turnover in K-free solution followed Arrhenius theory; the decline was linear from 37 to 4°C for all three groups. None showed a distinct break or transition temperature. Brading (1) also observed the K-insensitive 24Na turnover to be highly temperature dependent in tenia coli. The Q1o for the aortic tissues from the rat and squirrels were far from the value suggested for free diffusion. This implies that this exchange involved membrane-molecular interactions. It was postulated that K-stimulated sodium transport of squirrel cells would be less inhibited by cold than that of rat cells. At 8*C Na transport from both normothermic and hibernating squirrels was significantly greater than the rat. A distinct break occurred in the Arrhenius plot at 17°C for the rat aorta. A threefold increase in Qlo for active transport occurred below this temperature. Kstimulated sodium transport in squirrel vessels, however, followed Arrhenius theory over the 37.8*C temperature range. Because of the linear decline in rate, at lower temperatures the turnover exceeded that in the rat. This had adaptive significance for maintenance of ionic gradients in the cold. Furthermore, this adaptation was present in normothermic squirrels, which indicates it was
ZATZMAN,
JONES,
AND
SOUTH
not acquired by recent bouts of hypothermia. Kimzey and Willis (21) demonstrated that ouabain-sensitive sodium efflux from guinea pig erythrocytes was affected more by a decrease of temperature than was the sodium efflux from ground squirrel erythrocytes. This adaptation appears to be shared by excitable and nonexcitable tissues in the hibernator. In the present studies, K-stimulated Na transport fell linearly with temperature in the normothermic squirrel tissue, but fell off rapidly below 8*C in the hibernating squirrel. The implication of this observation is not clear. It seems unlikely that the membrane became “deadapted.” It is noted, however, that the low rate constant occurs below the temperature the animals attain when hibernating (20), and therefore may not affect the ability of the vessel to perform ionic transport during hibernation. Analysis of the effects of temperature on active transport were complicated by two problems. First, the rate of active Na efflux can only be estimated as a difference between total and inhibited unidirectional fluxes. Because of the insensitivity of the rat to ouabain (6) the Kdependent flux was used as an estimate of this function. This requires the [K]o surrounding the cells to be negligible during the K-free washout. This can be checked by estimating the maximum accumulation of K in the extracellular space. On return to 37°C the loss of K is about 1% per minute for tenia coli and myometrium (3, 12), which is equivalent to 1.5 mM fall in cell K during the first minute. Extracellular volume is twice intracellular (18); therefore in the case of no exchange, [K]o would increase by only 0.75 mM. However, diffusional delays in rat aorta of 140 ,um thickness (16) are small, having a half time of less than 10 s for 42K at 37*C (14). With more than 6 half times/min the [K]o at 37°C would be less than 10% of that above. According to the shape of the activation curve (15), [K]o would have to be in the 0.5 mM range to stimulate active transport. We assume then, that under the present conditions [K]o is not sufficiently high in K-free solutions to stimulate Na extrusion significantly. A second problem is that the tissues are no longer in a steady state when cooled (18). This is especially important for the rat. If during 40 min of washout at low temperature [Nalcell rises, then the rate constant at 37*C may not be an accurate reflection of the sodium efflux. The additional 40 min at 1°C was associated with a 75% increase in [Na],,u. The efflux at 37*C (rate x [Na]& wou ld therefore be gre tater than inferred fro m the plot of rate constants alone. Both measured and a corrected (1.75 x measured) value are included in Figs. 4 and 5 to give the maximum range. For low [NalCell, the rate should be rel .atively constant for small changes in [Nalcell, yielding a proportionality between [Na] cell and efflux (9, 15, 22). The measured value is probably the more reliable one. Although this factor limits detailed comparisons over the upper temperature range, the more critical comparisons at low temperatures are relatively unaffected because they were derived shortly after loading - in 24Na at 37*C. Aside from showing qu antitative differences in ionic tu rno,ver 1between groups, Arrhenius plots provide some
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (128.059.222.107) on January 15, 2019.
ION
TRANSPORT
IN
RAT
AND
GROUND
SQUIRREL
insight into the mechanism(s) by which aortic smooth muscle from the hibernator is adapted to support function at low temperature. Departures from linearity in Arrhenius plots have been interpreted in a number of ways. The effect of temperature on K-stimulated Na transport resulted in a monotonic decline in ionic turnover in the hibernator, while a break or increased E, occurred at 17°C in the nonhibernator. This may suggest that the phospholipids associated with the transport enzyme in the hibernator may be adapted to maintain fluidity at low temperatures. This postulate is supported by other findings of modifications in tissue lipid composition in hibernating mammals (10, 19, 24, 25). Implications can also be drawn from the differences between the effects of temperature on 42K turnover, Kfree 24Na turnover, and K-stimulated 24Na turnover. The Arrhenius plots for the rat vessels each have a unique configuration. One might conclude that the unidirectional fluxes occur at different sites in the membrane. Second, because the transition temperatures for 42K turnover (10°C) and K-stimulated 24Na turnover (17OC) are different, it is speculated that if these breaks in Arrhenius plots are due to phase changes in lipids, the phase change occurs only in the domain of the transport site. The domain concept is further supported by the observation that the 42K turnover is not in agreement with Arrhenius theory between 10°C and 0°C; turnover actually increases over this range. Lyons (23) postulated that a change in state could bring about a contraction of the membrane, causing cracks or channels to appear, which could lead to an increase in permeability. However, this seems unlikely since the loss of sodium does not increase at these temperatures. Finally, 24Na turnover in a K-free medium decreased linearly from 37OC to 4OC, in accord with Arrhenius theory. It seems probable that K-insensitive Na exchange occurs by ion exchange mechanisms separate from the Na-K pump. This is in agreement with the conclusions of Brading (1).
Flux Imbalance
c29
AORTA
at Low Temperature
An effort was made to synthesize the information on fluxes into a model to predict changes in ionic gradients at low temperature (Fig. 7). Measured K-stimulated Na efflux and rate constants for 42K washout were used to predict ionic fluxes on an hourly basis. The rate constants measured on tissues suddenly cooled to 7OC were assumed to be constant during prolonged cold. At least two factors may affect this assumption. One was the unknown effect of long-term cooling on membrane permeability, and the other was the increase in [Na],,n. Jones (15 and unpublished observations) has found the K-stimulated sodium efflux from the rat aorta to be proportional to [Nalcen over the lower range (12-25 mM). Saturation was approached above 25 mM. With cell sodium at low concentrations it was reasonable to assume that the rate constant remained invariant. The ratio of K efflux (assumed to be equal to K influx in steady state) and Kstimulated Na efflux at 37OC was unity in both the rat and squirrel tissues. It was assumed that this was also the case at 7OC and that the ratio remained unchanged during cold incubation. Finally, it was assumed that
Ground
Squirrel
Aorta
3Wa 1~1 coupling
Time
CKI cell
h-1
INaGe
1
3K pump
Hr-')
(.594
(fl/l/hr) 0
K lost Ic Na aained
a (.096
(fl)
Active Na efflux
K efflux
147
73
14x
140.6
19.4
13.49
Hr-') (rPl/l/hr)
7.72
6.4
11.52
2.0
2
138.6
21.4
13.31
12.71
.6
3
138.0
22.0
13.24
13.07
.2
4
137.8
22.2
13.23
13.19
.04
Theoretical
steady
state_
7Oc 4 hours
FIG. 7. Flux imbalance at 7OC in the ground squirrel aorta. Effluxes were calculated from the product of rate constants (h-‘) and intracellular ionic concentrations (Table 1, Ref. 18). The difference between active Na efflux (assumed equivalent to K influx) and K efflux was assumed equal to potassium lost per hour. This amount was subtracted from original cell K and added to cell Na. The subsequent fluxes were reiteratively calculated from the new ionic concentrations. When active Na efflux was equal to K efflux the cells were in a new steady state.
energy for transport was not limiting. Squirrel cells were in a “pump-leak” imbalance upon initial exposure to the cold. For approximately 5 K leaking out only 3 K were pumped in. Figure 7 predicts that as ionic concentrations change, the imbalance will decrease until a new steady state is achieved at 138 mM K and 22 mM Na, involving a shift of 10 mM Na and K during cold incubation (18). A direct check of the predictions were made by measuring slowly exchanging 24Na at 37 and 7OC. The increase in [Nalcell was complete by 6 h and stable up to 48 h at 7°C. Although the change in [Nalcell was only about one third that calculated in Fig. 7, the evidence supports the conclusion that the most important factor in bringing the tissues into a steady state at low temperature is the stimulation of active transport by elevated [Nalcell. Rat cells were also in a pump-leak imbalance on exposure to cold. For every 8 K leaking only 1 K was replaced. If all assumptions were correct, the rat tissue should have approached a new steady state of 105 mM K and 59 mM Na after 8 h in the cold. These levels were not in accord with observed values after 48 h of cold incubation (18). The discrepancy is attributable in part to saturation of the active transport mechanism before a pump-leak balance can be achieved. Other contributions
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c30
KAMM,
to the inability of these cells to retain ionic gradients might be a changing K permeability in the cold and/or inadequate metabolic support for transport. Additional experiments are needed before a quantitative description of pump-leak imbalance can be made for the rat. We thank Ms. Jeannette petent assistance and Dr. squirrels used in this study.
Kilfoil and Shu X. J. Musacchia
Hsien Liu Tsay for comfor donating the ground
ZATZMAN,
JONES,
AND
SOUTH
This work was supported by Public Health Service Grants HL17847, HL-07094, and HL-15852, and by the Research Council of the Graduate-School of the University of Missouri. A. W. Jones acknowledges the American Heart Association for his Established Investigator Fellowship. Present addresses: K. E. Kamm, Laboratorium voor Fysiologie, Campus Gasthuisberg, K.U.L., B-3000 Leuven, Belgium, and F. E. South, School of Life and Health Sciences, University of Delaware, Newark, DE 19717. Received
26 June
1978; accepted
in final
form
22 January
1979.
REFERENCES
BRADING, A. F. Sodium/sodium exchange in the smooth muscle of the guinea pig taenia coli. J. Physiol. London 251: 79-105, 1975. 2. BRADING, A., E. BULBRING, AND T. TOMITA. The effect of temper-
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ature on the membrane conductance of the smooth muscle of the guinea pig, taenia coli. J. Physiol. London 200: 621-635, 1969. CASTEELS, R., G. DROOGMANS, AND H. HENDRICKX. Membrane potential of smooth muscle cells in K-free solution. J. Physiol. London 217: 281-295, 1971. CASTEELS, R., G. DROOGMANS, AND H. HENDRICKX. Effect of sodium and sodium-substitutes on the active ion transport and on the membrane potential of smooth muscle cells. J. Physiol. London 228: 733-748, 1973. DANIEL, E. E. Potassium movements in rat uterus studied in vitro. I. Effects of temperature. Can. J. Biochem. Physiol. 41: 2065-2084, 1963. DANIEL, E. E. The interconnection between active transport and contractures in uterine tissues. Can. J. Biochem. Physiol. 42: 453495, 1964. ELFORD, B. C. Interactions between temperature and tonicity on cation transport in dog red cells. J. Physiol. London 246: 371-395, 1975. FREEMAN-NARROD, M., AND P. J. GOODFORD. Sodium and potassium content of the smooth muscle of the guinea-pig taenia coli at different temperatures and tensions. J. Physiol. London 163: 399410, 1962. GARRAHAN, P. J., AND R. P. GARAY. A kinetic study of the Na pump in red cells: its relevance to the mechanism of active transport. Ann. NY Acad. Sci. 242: 445-458, 1974. GOLDMAN, S. S. Cold resistance of the brain during hibernation. III. Evidence of a lipid adaptation. Am. J. Physiol. 228: 834-838,
1975. JONES, A. W. Factors
affecting sodium exchange and distribution myometrium. Physiol. Chem. Phys. 2: 79-95, 1970. 12. JONES, A. W. Effects of progesterone treatment on potassium accumulation and permeation in rabbit myometrium. Physiol. Chem. Phys. 2: 151-167, 1970. 13. JONES, A. W. Altered ion transport in vascular smooth muscle from spontaneously hypertensive rats: influence of aldosterone, norepinephrine and angiotensin. Circ. Res. 33: 563-572, 1973. 14. JONES, A. W. Analysis of bulk diffusion limited exchange of ions. In: Methods in Pharmacology. Smooth Muscle, edited by E. E. Daniel and D. M. Patton. New York: Plenum, 1975, chapt. 39, p. 673-687. in rabbit
15. JONES, A. W. K-stimulated Na transport in aortic smooth muscle and changes with DOCA hypertension in rats. Federation Proc. 35: 698, 1976. 16. JONES, A. W., P. D. SANDER, AND D. L. KAMPSCHMIDT. The effect of norepinephrine on aortic 42K turnover during deoxycorticosterone acetate hypertension and antihypertensive therapy in the rat. Circ. Res. 41: 256-260, 1977. 17. JONES, A. W., AND M. L. SWAIN. Chemical and kinetic analyses of sodium distribution in canine lingual artery. Am. J. Physiol. 223: 1110-1118, 1972. 18. KAMM, K. E., M. L. ZATAMAN, A. W. JONES, AND F. E. SOUTH. Maintenance of ion concentration gradients in the cold in aorta from rat and ground squirrel. Am. J. Physiol. 237: C17-C22, 1979 or Am. J. Physiol.: Cell Physiol. 6: Cl 7-C22, 1979. 19. KEITH, A. D., R. C. ALOIA, J. LYONS, W. SNIPES, AND E. T. PENGELLEY. Spin label evidence for the role of lysoglycerophosphatides in cellular membranes of hibernating mammals. Biochim. Biophys. Acta 394: 204-210, 1975. 20. KIMZEY, S. L., AND J. S. WILLIS. Resistance of erythrocytes of hibernating mammals to loss of potassium during hibernation and during cold storage. J. Gen. Physiol. 58: 620-633, 1971. 21. KIMZEY, S. L., AND J. S. WILLIS. Temperature adaptation of active sodium-potassium transport and of passive permeability in eryth’rocytes of ground squirrels. J. Gen. Physiol. 58: 634-649, 1971. 22. KNIGHT, A. B., AND L. G. WELT. Intracellular potassium: a determinant of the sodium-potassium pump rate. J. Gen. Physiol. 63: 351-373, 1974. 23. LYONS, J. Phase transitions and control of cellular metabolism at low temperatures. Cryobiology 9: 341-350, 1972. 24. MCMURCHIE, E. J., AND J. K. RAISON. Hibernation and homeothermic status of the Echidna (Tachyglossus aculeatus). J. Therm. BioZ. 1: 113-118, 1976. 25. RAISON, J. K., AND 3. M. LYONS. Hibernation: alteration of mitochondrial membranes as a requisite for metabolism at low temperature. Proc. Nat. Acad. Sci. USA 68: 2092-2094, 1971. 26. WILLIS, J. S. Characteristics of ion transport in kidney cortex of mammalian hibernators. J. Gen. Physiol. 49: 1221-1239, 1966. 27. WILLIS, J. S., L. S. T. FANG, AND R. F. FOSTER. The significance and analysis of membrane function in hibernation. In: Hibernation and Hypothermia, edited by F. E. South, J. P. Hannon, J. R. Willis, E. T. Pengelley, and N. R. Alpert. Amsterdam: Elsevier, 1972, p. 123-147.
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