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Effects of ions on thyrotrophin and prostaglandin E1 stimulation of glucose oxidation and adenyl cyclase-cyclic AMP system in dog thyroid slices
Effects of Ions on Thyrotrophin Stimulation
of Glucose Oxidation
and Prostaglandin
EI
and Adenyl Cyclase-Cyclic
AMP System in Dog Thyroid
Slices
By KAMEJIRO YAMASHITA, GAIL BLOOM, AND JAMES B. FIELD Substitution of K+ or choline for Na+ in Krebs-Ringer bicarbonate buffer increased basal glucose oxidation and 3H-adenine incorporation into sH-CAMP but had no consistent effect on CAMP concentrations. In high K+ buffer, TSH (3 mu/ml and 50 mu/ml) and prostaglandin E, (PGE,) (0.1 &g/ml and 10 pg/ml) had little or no effect on glucose oxidation while their stimulation of sH-CAMP formation and CAMP levels was equivalent to that obtained using regular KrebsRinger bicarbonate buffer. These results indicated that high K+ inhibited the effects of CAMP and not its generation. The increased basal glucose oxidation in high K+ buffer probably reflected increased membrane permeability since slices incubated with 14C-l-glucose and regular Krebs-Ringer bicarbonate buffer did not have augmented 14C02 production when they were later transferred to high K+ buffer. The increased sH-adenine incorporation into sH-CAMP cannot be explained by increased membrane per-
meability, but appeared to reflect an effect on the intracellular conversion of sH-adenine to sH-ATP or 3H-ATP to 3HCAMP. The effect of high K+ probably reflected low Na+ in the buffer since similar results were obtained when choline chloride was substituted for NaCl in Krebs-Ringer bicarbonate buffer. Exclusion of Ca f -t from Krebs-Ringer bicarbonate buffer decreased basal glucose oxidation, but had no effect on sH-adenine conversion to sH-CAMP or CAMP concentrations. Although effects of TSH and PGE, on glucose oxidation were diminished in buffer devoid of Ca++, their stimulation of 3H-CAMP formation and CAMP levels was similar in the presence and absence of Ca+ f. These results demonstrate that changes in the ionic composition of the buffer do not interfere with activation of the adenyl cyclaseCAMP system but modify some of the subsequent actions of the CAMP formed. (Metabolism 20: No.
A
LTERATION of the ionic concentration of the buffer has profound effects on various tissues incubated in vitro .I-* Increasing the K+ concentration stimulates hormonal secretion by the adrenal medulla,2 pituitary gland,3B4 and pancreas,5 and Ca++ ions appear to be essential for the secretion of insulin,5 steroid hormones,s LH,3 TSH,’ and prolacW in response to various stimuli. Previous studies have evaluated the effects of ions on several parameters of From the Clinical Research Unit and the Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pa. Received for publication December 28, 1970. This work was supported by USPHS Grant AM 06865 from The National Institutes of Health. KAMEJIRO YAMASHIW, M.D.: Research Associate, University of Pittsburgh School of Medicine, Pittsburgh, Pa. GAIL BLOOM, B.S.: Research Chemist, University of Pittsburgh School of Medicine, Pittsburgh, Pa. JAMES B. FIELD, M.D.: Professor of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pa. METABOLISM,
VOL. 20, No. 10 (OCTOBER), 1971
943
944
YAMASHITA, BLOOM, AND FIELD
thyroid function such as 32P incorporation into phospholipid,sJO glucose oxidation,11J2 colloid droplet formation,12 and cyclic 3’5’ adenosine monophosphate (CAMP) levels.12 Much current evidence suggests that CAMP mediates hormonal effects on the metabolism of target tissuesI and that most, if not all, of the effects of TSH and PGEl on the thyroid are secondary to increase CAMP accumulation.14-l7 The present studies were undertaken to elucidate further the effects of modifications of K+, Na+, and Ca++ concentrations of the buffer on TSH and PGE, stimulation of glucose oxidation and the adenyl cyclase-CAMP system. MATERIALS AND METHODS Dog thyroid glands were obtained, sliced, and incubated for determination of 1XZ-lglucose oxidation as previously described .16 Methods have been reported for measuring conversion of sH-adenine to sH-CAMP18 and the CAMP content19 of thyroid slices. High Kf (144 mM) Krebs-Ringer bicarbonate buffer was obtained by substituting equivalent amounts of isotonic KC1 for NaCl and 1.5% KHCO, for 1.3% NaHCO,. When effects of high K+ Krebs-Ringer bicarbonate buffer on 14C-l-glucose oxidation and CAMP concentrations were studied, slices were incubated in the appropriate buffer without preincubation for 45 and 20 min, respectively. When conversion of sH-adenine to aHCAMP was measured, slices were incubated for 1 hr at 37’C in 2 ml Krebs-Ringer bicarbonate or high K+ Krebs-Ringer bicarbonate buffer containing 1 mg/ml glucose and 10 pc sH-adenine. The slices were then rinsed in normal saline and incubated for another 30 min in 2 ml of the appropriate buffer containing 1 mg/ml glucose and 10-s it4 theophylline. TSH and PGE,, where appropriate, were only present during the second slices were incubated for 1 hr at incubation. In some experiments using I4C-l-glucose, 37OC in Krebs-Ringer bicarbonate buffer which also contained 0.1 gc 14C-l-glucose (100,000 cpm) and 1 mg/ml glucose. Following this incubation the slices were rinsed in normal saline and incubated for another 45 min in the appropriate buffer which contained unlabeled glucose (1 mg/ml). When TSH was present it was added only during the second incubation. Low Na+ Krebs-Ringer bicarbonate buffer was prepared by substituting isotonic choline was essentially zero since chloride for NaCl. In some experiments the Na + concentration KHCO, was used instead of NaHCO,. In other experiments NaHCO, was used resulting in a Na+ concentration of 23 m&4. In the experiments performed to study the effect of low Na+ buffer, the incubation conditions for measuring glucose oxidation, CAMP concentrations, and conversion of sH-adenine to sH-CAMP were the same as those described above for the experiments using high K+ buffer. Ca++-free Krebs-Ringer bicarbonate buffer was obtained by eliminating CaCl, from the buffer. In addition EGTA was added to the buffer to give a final concentration of 1O-3 M. The slices were preincubated in Ca ++-free and regular buffer as previously described.12 When sH-adenine incorporation into sH-CAMP was studied, 10 pc sH-adenine was added to the flask during the second incubation for 1 hr in either the Ca++-free or regular buffer. The slices were then rinsed and incubated for another 30 min in the appropriate buffer. During this last 30-min incubation the buffer contained 1 mg/ml glucose, 10-a M theophylline and the appropriate substance to be tested. The TSH and PGE, were dissolved as previously reported.12 RESULTS
of our previous results,12 incubation of thyroid slices in high Krebs-Ringer bicarbonate buffer caused marked stimulation of 14C-l-glucose oxidation. Under these conditions neither 3 nor 50 mu/ml TSH resulted in further 14C02 accumulation while both levels of TSH stimulated glucose oxidaIB confirmation
K+
EFFECTS
OF IONS ON GLUCOSE OXIDATION
:
ir
: i
3
e
3
e 1
945
946
YAMASHITA,
BLOOM, AND FIELD
tion in regular Krebs-Ringer bicarbonate buffer (Table 1). Thyroid slices incubated in high K+ buffer incorporated significantly more 3H-adenine into 3H-CAMP than controls incubated in Krebs-Ringer bicarbonate buffer. In contrast to the results measuring glucose oxidation, both 3 and 50 mu/ml TSH significantly increased 3H-adenine incorporation into “H-CAMP. The percent stimulation by TSH was about the same in the slices incubated in high K+ and regular Krebs-Ringer bicarbonate buffer. CAMP levels were not consistently different using either high K+ or regular Krebs-Ringer bicarbonate buffer’? and the increase due to TSH was essentially the same in both buffers. The results in Table 2 demonstrate similar findings when PGE, was used instead of TSH. Basal glucose oxidation was greater in high K+ buffer and effects of PGEl were minimal as compared to slices incubated in regular Krebs-Ringer bicarbonate buffer. High Kf buffer again increased “H-adenine conversion to 3H-CAMP. Basal CAMP was the same in both buffers, and, although PGE, tended to have a greater effect in high K+ buffer. this was not always the case. The studies summarized in Table 3 were done in an attempt to dissociate an effect of increased K+ on glucose transport from an effect on intracellular glucose metabolism. The ability of high KC buffer to augment glucose oxidation is again evident from the experiments in which only a single 45min incubation was done. However, when the thyroid slices were incubated in regular Krebs-Ringer bicarbonate buffer containing 14C-l-glucose for 1 hr to label the intracellular precursors of 14C02 and the slices were rinsed and incubated with unlabeled glucose, basal 14C0., production was the same in both high K+ and regular Krebs-Ringer bicarbonate buffer. TSH ( 5 mu/ml) significantly increased 14C-lglucose oxidation when the second incubation was done in regular and high K+ Krebs-Ringer bicarbonate buffer. The difference in the basal glucose oxidation in the experiment utilizing a single incubation as compared to a preincubation with lSC-l-glucose reflects several factors. In the latter studies much of the of Preincubation of Dog Thyroid Slices in Krebs-Ringer Bicarbonate Table 3 .-Effect Buffer on Basal and TSH Stimulated 14C-l-Glucose Oxidation * Krebs-Ringer Experimental conditions
Single 45min incubation 1-hr preincubation with 14C-lglucose Single 45min incubation 1-hr preincubation with 14C-lglucose
Bicarbonate
‘“COz-produced (c m/‘a) Buffer Hiah f;+ Krebs-Ringer
Control
TSH (SmU/ml)
43,700?2,800
100,300~4,700
4,400t
200
74,000?4,100
3.900*
300
10,100-c
138,2OO-t9,300
8,300-c
400
82.800 + 2,500
4,400*
200
158.500~10,800
4,800?
Buffer
TSH (SmU/ml)
Control
800
Bicarbonate
200
104,100+- 2.100
9,OOOk
300
173,900+ 13,700
8,300+
400
* The results are the average k SEM of triplicate determinations. Two separate experiments are shown. Slices from the same dog thyroid were used in each experiment. When slices were preincubated with 14C-l-glucose, unlabeled glucose (1 mg/ml) was present during that and the subsequent 45min incubation period. TSH was present only during the second incubation period.
947
EFFECTS OF IONS ON GLUCOSE OXIDATION
14C-l-glucose would have been oxidized to l”COZ and not collected during the first hour of incubation. During the subsequent 45 min of incubation when l*COZ was collected, unlabeled glucose was present in the buffer and this would dilute the specific activity of the precursors of the 14C02 in the cells. Although these results suggests an effect of high K+ to increase glucose transport into the cell, studies utilizing 3H-adenine indicate the effects of K+ are more complicated. Initial incubation of thyroid slices with 3H-adenine and either high K+ or regular Krebs-Ringer bicarbonate buffer, followed by incubation in regular Krebs-Ringer buffer, resulted in formation of similar amounts of 3H-CAMP (Table 4). However, when the second incubation was done using high K+ buffer, 3H-CAMP formation was significantly greater, whether high K+ or regular Krebs-Ringer buffer was used during the first incubation. In general, conversion of 3H-adenine to 3H-CAMP was greater when both the first and second incubations were done with high K+ buffer. In these experiments, effects of TSH and PGEl were very similar to those shown in Tables 1 and 2. In the latter experiments the initial and second incubations were done in the same buffer. Since the high K+ buffer was also low in Na +, the results could reflect changes in either ion. To evaluate more directly the effects of low Na+, choline chloride was substituted for NaCl (Table 5). In some of the experiments the Na+ was totally replaced because KHCO, was used instead of NaHCO.?. Basal glucose oxidation was markedly stimulated with very little additional effect from 3 mu/ml TSH when Na+ was reduced in the buffer. This result was similar to that obtained with high K+ buffer. Incorporation of 3H-adenine into 3H-CAMP and CAMP levels was similar in both buffers, and the response to TSH was very similar. Previously we had reported that exclusion of Ca++ from Krebs-Ringer bicarbonate buffer reduced basal glucose oxidation and the response to a low but not a high dose of TSH. l1 The results in Table 6 indicate that Ca++ is not necessary for TSH stimulation of 3H-adenine conversion to 3H-CAMP or elevation of CAMP concentrations, but is essential for TSH stimulation of glucose oxidation when 3 mu/ml of the hormone is used. In the absence of TSH, exclusion of Ca++ from the buffer did not modify either 3H-CAMP formation or Table 4.-Effect of K+ Concentration of Krebs-Ringer Bicarbonate Buffer on TSH and PGEl Stimulation of 3H-Adenine Incorporation Into 3H-CAMP in Dog Thyroid Slices * Incubation
Buffer
“H-CAMP TSH
First
Second
Regular
Regular
High
High
K+
Regular
High
K+
High
Regular
K+ K+
Control 5.500 20,200
f
(3 mu/ml) 800
(cpm/g) TSH
(50 mU/ml)
PGEl
PGEI
(1 !Jg/mlj
(10 Irg/ml)
17,000
+ 1,200
33,200
f
500
23,600
+
400
27,000
k 4,100
30,200
5
35,500 f
700
29,700
+ 1,300
58.700
+ 2,100
2 5,900
9,000
f
1,400
4,300
5
500
800
* The results are the average & SEM of triplicate determinations. The first incubation was for 1 hr in the appropriate buffer which contained 10 PC sH-adenine and 1 mg/ml glucose. The slices were then rinsed and incubated for 30 min in the appropriate buffer containing 10-s M theophylline and the substance to be tested. The results are representative of six different experiments.
948
YAMASHITA, BLOOM, AND FIELD
Table K-Effect of Absence of Na+ on TSH Stimulation of 14C-l-Glucose Oxidation, CAMP Concentrations, and %Adenine Incorporation into sH-CAMP in Dog Thyroid Slices * Low Na+ Kreb;;Eyr Krebs-Ringer Control
Experiment
Bicarbonate
Bicarbonate
Buffer
TSH (3 mU/ml)
Control
TSH (3 mu/ml)
1
14C0, cpm/g sH-CAMP cpm/g CAMP mm/g Experiment 2 14COs cpm/g sH-CAMP cpm/g CAMP wm/g
39,900 + 2,600 12,500+ 1,100 1.550.2
60,200-t 1,000 22,500a 1,600 3.6kO.3
101,800+ 300 17,300+ 1,200 1.7kO.l
110,400c3,800 32,100? 1,400 3.OkO.2
35,10012,400 10,400t 700 1.220.1
72,6OO-c3,100 30,600” 900 1.820.1
79,600?4,800 10,700-1- 1,100 l.l+-0.1
92,600r5,500 46,600+ 1,300 2.5t0.3
* The results are the average ? SEM of triplicate determinations for glucose oxidation and sH-adenine incorporation into sH-CAMP and quadruplicate determinations for CAMP concentrations. In the low Na+ Krebs-Ringer bicarbonate buffer, the NaCl was replaced with isotonic choline chloride. In Experiment 1, KHCO, was used instead of NaHCO,. In Experiment 2, NaHCO, was used to make the low Nat- Krebs-Ringer bicarbonate buffer.
levels of CAMP. In Ca++-free buffer, 0.1 pg/ml PGE1, as 3 mu/ml TSH, did not increase 14C02 formation (Table 7). Increasing the PGEl to 10 pg/ml, a maximum dose,12 caused stimulation of glucose oxidation, but the effect was less than that produced in buffer containing Ca ++. Despite the decreased effects of PGEl on glucose oxidation, both concentrations of PGEl caused significant increases in 3H-adenine conversion to 3H-CAMP and CAMP concentrations. Although basal values for 3H-CAMP formation and CAMP concentrations were sometimes lower in Ca++-free buffer, the per cent stimulation induced by PGEl was usually the same as in regular Krebs-Ringer bicarbonate buffer. DISCUSSION
The present data confirm our previous results that a high K+ concentration in the buffer mimics the effects of TSH and increases glucose oxidation.12 In those experiments there were no consistent effects of high K+ on CAMP concentration in thyroid slices, and it was postulated that the enhanced 14C02 production reflected changes in glucose transport rather than activation of the adenyl cyclaseCAMP system. Support for an effect of high K+ on membrane transport of glucose was provided by the experiments in which intracellular precursors of 14COn ‘. were labeled prior to incubating thyroid slices in high K+ buffer. Under these conditions (Table 3) basal glucose oxidation was not increased by high K+ buffer, a result which would be anticipated if the primary effect of high K+ were to increase transport of glucose from the medium into the cell. Such an effect of K+ on membrane permeability would be consistent with the observations of Williams who measured membrane potential in thyroid cells.20 He observed that increasing K+ in the buffer was associated with decreasing negativity of the membrane potential. Dekker and Field provided histologic and electron microscopic evidence of morphologic changes in thyroid slices induced by high K+ buffer that would be compatible with alterations in membrane transport.12
Bicarbonate TSH (50 mu/ml)
216,400 e 11,600 14,400 t 2,700 4.9 32.0 +
Buffer
cpm/g
34,400 + 1,200 4,200 t 400 4.9 + 0.7
Control
23,900 t 500 2,300 c 200 1.5 + 0.1
59,700 4 1,000 19,400 & 800 2.1 15.8 t
46,900 + 1,600 11,900 2 1,600 7.6 e 0.8
35,500 f 1,200 8,500 + 500 2.8 -c 0.3
incorporation
into aHCAMP
and quad-
31,000 + 1,000 100 14,000 f 0.5 4.9 2
25,400 t 1,200 4,000 * 100 2.6 f 0.4
Buffer
and
PGEx (10 Irg/mU
Bicarbonate
Concentration
and quad-
PGEi (0.1 wg/ml)
Krebs-Ringer
and
77,200 k 1,000 20,800 k 1,600 5.8 22.7 f into sH-CAMP
CAMP
incorporation
33,900 + 400 8,400 + 600 10.7 +- 2.5
TSH (50 mu/d)
Buffer
AMP Concentration Bicarbonate
TSH (3 mu/ml)
Oxidation, Slices * Cat+-free
Control
Buffer
Control
Bicarbonate
of 14C-l-Glucose in Dog Thyroid
PGEl (10 W/ml)
Krebs-Ringer
of Ca++ From Buffer on PGEl Stimulation 3H-Adenine Incorporation Into 3H-CAMP PGEI (0.1 ~gg/d)
of Exclusion
* The results are the average k SEM of triplicate determinations for glucose oxidation and sH-adenine ruplicate determinations for CAMP concentrations and are representative of three different experiments.
aHCAMP cpm/g CAMP mpm/g
wo,
Table 7.-Effect
Cyclic
Ca++-free Krebs-Ringer
* The results are the average + SEM of triplicate determinations for glucose oxidation and sH-adenine ruplicate determinations for CAMP concentrations and are representative of six different experiments.
76,700 c 1,900 8,300 + 200 8.1 t 1.3
Krebs-Ringer
of Ca+ + From Buffer on TSH Stimulation of ‘4C-l-Glucose Oxidation, 3H-Adeuine Incorporation Into 3H-CAMP in Dog Thyroid Slices *
47,000 +- 1,700 4,200 c 300 4.0 + 0.4
of Exclusion
sH-CAMP cpm/g CAMP mum/g
14cq? cpm/g
Table 6.Effect
950
YAMASHITA,
BLOOM, AND FIELD
However, the role of K+ is certainly more complex than simply altering glucose transport into the cell. Previously we had demonstrated that insulin augmented glucose uptake by thyroid slices but did not stimulate glucose oxidation.?’ The data in Table 4 do not indicate an effect of high K+ on 3H-adenine uptake into the cell. Basal 3H-CAMP formation was essentially the same when high K+ and regular Krebs-Ringer bicarbonate buffers were used during the incubation with 3H-adenine and a regular buffer used for the subsequent incubation. Examination of the results obtained when slices were incubated in regular Krebs-Ringer bicarbonate buffer and 3H-adenine and then transferred to either regular or high K+ buffer clearly indicates that K+ in the second incubation augmented 3H-CAMP formation. This could reflect either an effect of K+ on “H-ATP formation from 3H-adenine or increased :$H-ATP conversion to “HCAMP. The absence of a clear-cut stimulation of CAMP concentrations by high K+l” militates against the second possibility. This is different from the results reported in other tissues. High K+ in the buffer increased CAMP concentrations in parotid gland,“” brain,“” and rat diaphragm.2J The apparent higher specific activity of the CAMP in the presence of K+ would be compatible with increased incorporation of “H-adenine into 3H-ATP. In these experiments, the high K+ buffer was also deficient in Na+, raising the possibility that the results reflected absence of this iron rather than surfeit of K+. The results in Table 5 indicate that the reduction in Na+ is probably more important than the excess of K+ since replacement of NaCl with choline chloride also increased glucose oxidation but decreased TSH stimulation of l:‘CO,. The effect of TSH on 3H-adenine conversion to 3H-CAMP and CAMP concentrations was similar in buffer with normal and reduced amounts of Na+. This is again consistent with the concept that alterations in ionic composition of the buffer modify effects of CAMP and not its generation. The role of Ca++ in mediating the effects of TSH on various aspects of thyroid function has also been examined. Zor et al. reported that basal glucose oxidation was decreased in thyroid slices incubated in the absence of Ca++ and that the TSH (3 mu/ml) stimulation of W02 production and 32P incorporation into phospholipid was inhibited .ll However, increasing the concentration of TSH to 50 mu/ml appeared to overcome the inhibition. Since the effects of the dibutyryl derivative of CAMP were also reduced in the absence of Ca++, it was suggested that Ca++ was essential for the action of the cyclic nucleotide rather than for its formation. Dekker and Field provided additional support for this conclusion since elimination of Ca ++ from the buffer did not diminish the TSH increase in CAMP.l” The dose of TSH was ineffective in stimulating glucose oxidation in the absence of Ca ++. Previously Bakke et al. had reported that Ca++ was essential for the action of TSH on increasing thyroid slice weight.“’ Ca++ also appeared to be necessary for incorporating Is11 into thyroglobulin.2G In contrast to these effects, TSH was still capable of stimulating colloid droplet formation in the absence of Ca ++.I2 Thus, there is a dissociation between Ca++ dependence and the various actions of CAMP. The data in Table 6 confirm the results that Ca++ is not essential for TSH stimulation of the adenyl cyclase-CAMP system. Both the low and the high doses of TSH caused equivalent increments in CAMP levels and 3H-adenine conversion
EFFECTS
OF IONS ON GLUCOSE OXIDATION
951
to 3H-CAMP whether Ca++ was present in the buffer or not. These results are somewhat at variance with those obtained when the adenyl cyclase activity of homogenates has been measured. Burke reported that Ca++ decreased basal adenyl cyclase activity and inhibited the stimulation induced by TSH and F- in homogenates of sheep thyroid glands. 27 Bar and Hechter observed that EGTA increased basal adenyl cyclase activity in mitochondrial and microsomal fractions of bovine adrenal cortex and rat fat cell ghosts but abolished stimulation caused by ACTH.28 Bradham et al. found that EGTA inhibition of adenyl cyclase activity of homogenates of calf cerebral cortext could be reversed by addition of Ca++.3” In contrast to our results, Kuo noted that TSH stimulation of 3H-adenine incorporation into 3H-CAMP in isolated adipose cells was dependent upon Ca++.30 In his experiments Ca++ was also essential for the action of ACTH and LH but not for norepinephrine and glucagon. Such differences were interpreted as reflecting differences in the binding of the hormones to the cell receptor and their subsequent activation of adenyl cyclase. There is no compelling reason why the receptor sites of one tissue might require Ca++ while those of another might not even though the same hormone was involved in the stimulation. Certainly the effects of TSH in thyroid appear to be much more specific than on adipose tissue. Experiments utilizing intact cells in tissue slices are probably more meaningful than those measuring effects of ions on adenyl cyclase in tissue homogenates. An excellent example of this is the dissociation of the effects of Fon the adenyl cyclase-CAMP system in thyroid homogenate and intact cells in slicesI The mechanism by which Ca++ is essential for the expression of effects of CAMP is not known. This problem as well as the relationship between Ca++ and hormone action was recently reviewed by Rasmussen.3r Studies were done using PGE, and TSH in hopes that additional information would be obtained related to the mechanism of their action on various parameters of thyroid function. Although PGEl mimicked many of the effects of TSH on the adenyl cyclase-CAMP system, on glucose oxidation, and 32P incorporation into phospholipids, some of the results suggested that its action might be mediated by a mechanism different from that of TSH.32 In general, the effects of PGE, in high K+ buffer were similar to those obtained using TSH. Although PGE, caused greater stimulation of CAMP levels in slices incubated in high K+ buffer (Table 2), this was not a consistent finding and did not indicate greater responsiveness of adenyl cyclase activity to PGE, under these circumstances. These results, and those with TSH, indicate that high K+ interfered with the action of CAMP and not its generation induced by PGE,. The results obtained with PGEl in Ca ++-free buffer were also qualitatively similar to those observed with TSH. The low dose of PGEl (0.1 Irg/ml) was completely ineffective in increasing glucose oxidation while a higher dose (10 pg/ml) caused a significant effect. Although 0.1 pg/ml PGEl did not increase 14C02 formation, it still stimulated 3H-adenine incorporation into 3H-CAMP and CAMP concentration, further supporting the role of Ca++ in the expression of the effects of CAMP rather than its generation. The apparent reduction of PGE, stimulation of adenyl cyclase-CAMP system in high K+ buffer (Table 7) was not consistently found in all experiments. The absence of clear-cut differ-
952
YAMASHITA, BLOOM, AND FIELD
ences between the responses to TSH and PGEl in high K+ or Ca++-fkee buffers does not necessarily indicate that they produce these changes by the same mechanism or that they stimulate adenyl cyclase activity by the same receptors. ACKNOWLEDGMENT We are indebted to Mrs. Loretta Malley and Miss Barbara Sheehan for expert secretarial assistance. Bovine TSH (2 U/mg) was a generous gift from the Endocrinology Study Section, National Institutes of Health. Prostaglandin E, was kindly provided by Dr. John Pike of the Upjohn Company, Kalamazoo, Mich. 14C-l-glucose (2.4 mCi/mmole) was purchased from Amersham-Searle Corporation. sH-adenine (22 Ci/mmole) and aH-CAMP (2.35 Ci/mmole) were obtained from Schwartz Bio-Research Corporation. REFERENCES 1. Douglas, W. W.: A possible mechanism of neurosecretion: Release of vasopressin by depolarization and its dependence on calcium. Nature 197:81, 1963. 2. -, and Rubin, R. P.: The role of calcium in the secretory response of the adrenal medulla to acetylcholine. J. Physiol. 159:40, 1961. 3. Samli, M. H., and Geschwind, I. I.: Some effects of energy-transfer inhibitors and of Ca++-free or K+-enhanced media on the release of luteinizing hormone (LH) from the rat pituitary gland in vitro. Endocrinology 82:225, 1968. 4. Kraicer, J., Milligan, J. V., Gosbee, J. L., Conrad, R. G., and Branson, C. M.: In vitro release of ACTH: Effects of potassium, calcium and corticosterone. Endocrinology 85: 1144, 1969. 5. Lambert, A. E., Jeanrenaud, B., Junod, A., and Renold, A. E.: Organ culture of fetal rat pancreas. II. Insulin release induced by amino and organic acids, by hormonal peptides, by cationic alterations of the medium and by other agents. Biochim. Biophys. Acta 184:540, 1969. 6. Birmingham, M. K., Elliott, F. H., and Valere, P. H.-L.: The need for the presence of calcium for the stimulation in vitro of rat adrenal glands by adrenocorticotrophic hormone. Endocrinology 53:687, 1953. 7. Vale, W., Burgus, R., and Guillemin, R.: Presence of calcium ions as a requisite for the in vitro stimulation of TSH-release by hypothalamic TRF. Experientia 23:853, 1967. 8. Parsons, J. A.: Calcium ion requirement for prolactin secretion by rat adenohypophyses in vitro. Amer. J. Physiol. 217: 1599, 1969. 9. Oka, H., and Field, J. B.: Effects of ions on TSH stimulation of s*P incorpora-
tion into thyroid slice phospholipid. Amer. J. Physiol. 211:1357, 1966. 10. Burke, G.: Effects of thyrotropin, sodium fluoride and ions on thyroid slice metabolism. Metabolism 19:35, 1970. 11. Zor, U., Lowe, I. P., Bloom, G., and Field, J. B.: The role of calcium (Ca++) in TSH and dibutyryl 3’5’-cyclic AMP stimulation of thyroid glucose oxidation and phospholipid synthesis. Biochem. Biophys. Res. Commun. 33:649, 1968. 12. Dekker, A., and Field, J. B.: Correlation of effects of thyrotropin, prostaglandins and ions on glucose oxidation, cyclic-AMP, and colloid droplet formation in dog thyroid slices. Metabolism 19:453, 1970. 13. Sutherland. E. W., and Robison, G. A.: The role of cyclic-3’5’-AMP in responses to catcholamines and other hormones. Pharmacol. Rev. 18:145, 1966. 14. Pastan, I., and Katzen, R.: Activation of adenyl cyclase in thyroid homogenates by thyroid-stimulating hormone. Biochem. Biophys. Res. Commun. 29:792, 1967. 15. Zor, U., Kaneko, T., Lowe, I. P., Bloom, G.. and Field, J. B.: Effect of thyroid-stimulating hormone and prostaglandins on thyroid adenyl cyclase activation and cyclic adenosine 3’5’-monophosphate. J. Biol. Chem. 244:5189, 1969. 16. Gilman, A. G., and Rail, T. W.: The role of adenosine 3’5’-phosphate in mediating effects of thyroid-stimulating hormone on carbohydrate metabolism of bovine thyroid slices. I. Biol. Chem. 243:5872, 1968. 17. Kaneko, T., Zor, U., and Field, J. B.: Thyroid stimulating hormone and prostaglandin Et stimulating of cyclic 3’5’-adenosine monophosphate in thyroid slices. Science 163: 1062, 1969. 18. -, -, and -: Stimulation of thyroid adenyl cyclase activity and cyclic adenosine
EFFECTS
OF IONS ON GLUCOSE
OXIDATION
3’5’-monophosphate by long-acting thyroid stimulator. Metabolism 19:430, 1970. 19. -, and Field, J. B.: A method for determination of 3’5’cyclic adenosine monophosphate based on adenosine triphosphate formation. J. Lab. Clin. Med. 74:682, 1969. 20. Williams, J. A.: Effect of external K+ concentration on transmembrane potentials of rabbit thyroid cells. Amer. I. Physiol. 211:1171, 1966. 21. Field, J. B., Pastan, I., Johnson, P., and Herring, B.: Stimulation in vitro of pathways of glucose oxidation in thyroid by thyroid stimulating hormone. J. Biol. Chem. 235: 1863, 1960. 22. Rasmussen, H., and Tenenhouse, A.: Cyclic adenosine monophosphate, Ca+ +, and membranes. Proc. Nat. Acad. Sci. USA 59:1364, 1968. 23. Sattin, A., and Rall, T. W.: The effect of brain extracts on the accumulation of cyclic 3’5’-AMP (CA) in slices of guinea pig (GP) cerebral cortex. Fed. Proc. 26: 707, 1967. 24. Ludholm, L., Rall, T., and Vamos, N.: Influence of K-ions and adrenaline on the adenosine 3’S’-monophosphate content in rat diaphragm. Acta Physiol. Stand. 70: 127, 1967. 25. Bakke, J. L., Heideman, M. L., Lawrence, N. L., and Wiberg, C.: Bioassay of thyrotropic hormone by weight response of bovine thyroid slices. Endocrinology 61:352,
953 1957. 26. Kondo, Y., and Ui, N.: A new assay method for thyrotropic hormone based on the iodination of thyroglobulin in hog thyroid slices. Endocr. Jap. 10:60, 1963. 27. Burke, G.: Effects of cations and ouabain on thyroid adenyl cyclase. Biochim. Biophys. Acta 220:30, 1970. 28. Bar, H.-P., and Hechter, 0.: Adenyl cyclase and hormone action. III. Calcium requirement for ACTH stimulation of adenyl cyclase. B&hem. Biophys. Res. Commun. 35:681, 1969. 29. Bradham, L. S., Holt, D. A., and Sims, M.: The effect of Ca++ on the adenyl cyclase of calf brain. B&him. Biophys. Acta 201:250, 1970. 30. Kuo, J. F.: Differential effects of Ca++, EDTA and adrenergic blocking agents on the actions of some hormones on adenosine 3’5’-monophosphate levels in isolated adipose cells as determined by prior labeling with (8-1X!) adenine. Biochim. Biophys. Acta 208:509, 1970. 3 1. Rasmussen, H.: Cell communication, calcium ion, and cyclic adenosine monophosphate. Science 170:404, 1970. 32. Zor, U., Bloom, G., Lowe, I. P., and Field, J. B.: Effects of theophylline, prostaglandin E, and adrenergic blocking agents on TSH stimulation of thyroid intermediary metabolism. Endocrinology 84: 1082, 1969.
of Glucose Oxidation
and Prostaglandin
EI
and Adenyl Cyclase-Cyclic
AMP System in Dog Thyroid
Slices
By KAMEJIRO YAMASHITA, GAIL BLOOM, AND JAMES B. FIELD Substitution of K+ or choline for Na+ in Krebs-Ringer bicarbonate buffer increased basal glucose oxidation and 3H-adenine incorporation into sH-CAMP but had no consistent effect on CAMP concentrations. In high K+ buffer, TSH (3 mu/ml and 50 mu/ml) and prostaglandin E, (PGE,) (0.1 &g/ml and 10 pg/ml) had little or no effect on glucose oxidation while their stimulation of sH-CAMP formation and CAMP levels was equivalent to that obtained using regular KrebsRinger bicarbonate buffer. These results indicated that high K+ inhibited the effects of CAMP and not its generation. The increased basal glucose oxidation in high K+ buffer probably reflected increased membrane permeability since slices incubated with 14C-l-glucose and regular Krebs-Ringer bicarbonate buffer did not have augmented 14C02 production when they were later transferred to high K+ buffer. The increased sH-adenine incorporation into sH-CAMP cannot be explained by increased membrane per-
meability, but appeared to reflect an effect on the intracellular conversion of sH-adenine to sH-ATP or 3H-ATP to 3HCAMP. The effect of high K+ probably reflected low Na+ in the buffer since similar results were obtained when choline chloride was substituted for NaCl in Krebs-Ringer bicarbonate buffer. Exclusion of Ca f -t from Krebs-Ringer bicarbonate buffer decreased basal glucose oxidation, but had no effect on sH-adenine conversion to sH-CAMP or CAMP concentrations. Although effects of TSH and PGE, on glucose oxidation were diminished in buffer devoid of Ca++, their stimulation of 3H-CAMP formation and CAMP levels was similar in the presence and absence of Ca+ f. These results demonstrate that changes in the ionic composition of the buffer do not interfere with activation of the adenyl cyclaseCAMP system but modify some of the subsequent actions of the CAMP formed. (Metabolism 20: No.
A
LTERATION of the ionic concentration of the buffer has profound effects on various tissues incubated in vitro .I-* Increasing the K+ concentration stimulates hormonal secretion by the adrenal medulla,2 pituitary gland,3B4 and pancreas,5 and Ca++ ions appear to be essential for the secretion of insulin,5 steroid hormones,s LH,3 TSH,’ and prolacW in response to various stimuli. Previous studies have evaluated the effects of ions on several parameters of From the Clinical Research Unit and the Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pa. Received for publication December 28, 1970. This work was supported by USPHS Grant AM 06865 from The National Institutes of Health. KAMEJIRO YAMASHIW, M.D.: Research Associate, University of Pittsburgh School of Medicine, Pittsburgh, Pa. GAIL BLOOM, B.S.: Research Chemist, University of Pittsburgh School of Medicine, Pittsburgh, Pa. JAMES B. FIELD, M.D.: Professor of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pa. METABOLISM,
VOL. 20, No. 10 (OCTOBER), 1971
943
944
YAMASHITA, BLOOM, AND FIELD
thyroid function such as 32P incorporation into phospholipid,sJO glucose oxidation,11J2 colloid droplet formation,12 and cyclic 3’5’ adenosine monophosphate (CAMP) levels.12 Much current evidence suggests that CAMP mediates hormonal effects on the metabolism of target tissuesI and that most, if not all, of the effects of TSH and PGEl on the thyroid are secondary to increase CAMP accumulation.14-l7 The present studies were undertaken to elucidate further the effects of modifications of K+, Na+, and Ca++ concentrations of the buffer on TSH and PGE, stimulation of glucose oxidation and the adenyl cyclase-CAMP system. MATERIALS AND METHODS Dog thyroid glands were obtained, sliced, and incubated for determination of 1XZ-lglucose oxidation as previously described .16 Methods have been reported for measuring conversion of sH-adenine to sH-CAMP18 and the CAMP content19 of thyroid slices. High Kf (144 mM) Krebs-Ringer bicarbonate buffer was obtained by substituting equivalent amounts of isotonic KC1 for NaCl and 1.5% KHCO, for 1.3% NaHCO,. When effects of high K+ Krebs-Ringer bicarbonate buffer on 14C-l-glucose oxidation and CAMP concentrations were studied, slices were incubated in the appropriate buffer without preincubation for 45 and 20 min, respectively. When conversion of sH-adenine to aHCAMP was measured, slices were incubated for 1 hr at 37’C in 2 ml Krebs-Ringer bicarbonate or high K+ Krebs-Ringer bicarbonate buffer containing 1 mg/ml glucose and 10 pc sH-adenine. The slices were then rinsed in normal saline and incubated for another 30 min in 2 ml of the appropriate buffer containing 1 mg/ml glucose and 10-s it4 theophylline. TSH and PGE,, where appropriate, were only present during the second slices were incubated for 1 hr at incubation. In some experiments using I4C-l-glucose, 37OC in Krebs-Ringer bicarbonate buffer which also contained 0.1 gc 14C-l-glucose (100,000 cpm) and 1 mg/ml glucose. Following this incubation the slices were rinsed in normal saline and incubated for another 45 min in the appropriate buffer which contained unlabeled glucose (1 mg/ml). When TSH was present it was added only during the second incubation. Low Na+ Krebs-Ringer bicarbonate buffer was prepared by substituting isotonic choline was essentially zero since chloride for NaCl. In some experiments the Na + concentration KHCO, was used instead of NaHCO,. In other experiments NaHCO, was used resulting in a Na+ concentration of 23 m&4. In the experiments performed to study the effect of low Na+ buffer, the incubation conditions for measuring glucose oxidation, CAMP concentrations, and conversion of sH-adenine to sH-CAMP were the same as those described above for the experiments using high K+ buffer. Ca++-free Krebs-Ringer bicarbonate buffer was obtained by eliminating CaCl, from the buffer. In addition EGTA was added to the buffer to give a final concentration of 1O-3 M. The slices were preincubated in Ca ++-free and regular buffer as previously described.12 When sH-adenine incorporation into sH-CAMP was studied, 10 pc sH-adenine was added to the flask during the second incubation for 1 hr in either the Ca++-free or regular buffer. The slices were then rinsed and incubated for another 30 min in the appropriate buffer. During this last 30-min incubation the buffer contained 1 mg/ml glucose, 10-a M theophylline and the appropriate substance to be tested. The TSH and PGE, were dissolved as previously reported.12 RESULTS
of our previous results,12 incubation of thyroid slices in high Krebs-Ringer bicarbonate buffer caused marked stimulation of 14C-l-glucose oxidation. Under these conditions neither 3 nor 50 mu/ml TSH resulted in further 14C02 accumulation while both levels of TSH stimulated glucose oxidaIB confirmation
K+
EFFECTS
OF IONS ON GLUCOSE OXIDATION
:
ir
: i
3
e
3
e 1
945
946
YAMASHITA,
BLOOM, AND FIELD
tion in regular Krebs-Ringer bicarbonate buffer (Table 1). Thyroid slices incubated in high K+ buffer incorporated significantly more 3H-adenine into 3H-CAMP than controls incubated in Krebs-Ringer bicarbonate buffer. In contrast to the results measuring glucose oxidation, both 3 and 50 mu/ml TSH significantly increased 3H-adenine incorporation into “H-CAMP. The percent stimulation by TSH was about the same in the slices incubated in high K+ and regular Krebs-Ringer bicarbonate buffer. CAMP levels were not consistently different using either high K+ or regular Krebs-Ringer bicarbonate buffer’? and the increase due to TSH was essentially the same in both buffers. The results in Table 2 demonstrate similar findings when PGE, was used instead of TSH. Basal glucose oxidation was greater in high K+ buffer and effects of PGEl were minimal as compared to slices incubated in regular Krebs-Ringer bicarbonate buffer. High Kf buffer again increased “H-adenine conversion to 3H-CAMP. Basal CAMP was the same in both buffers, and, although PGE, tended to have a greater effect in high K+ buffer. this was not always the case. The studies summarized in Table 3 were done in an attempt to dissociate an effect of increased K+ on glucose transport from an effect on intracellular glucose metabolism. The ability of high KC buffer to augment glucose oxidation is again evident from the experiments in which only a single 45min incubation was done. However, when the thyroid slices were incubated in regular Krebs-Ringer bicarbonate buffer containing 14C-l-glucose for 1 hr to label the intracellular precursors of 14C02 and the slices were rinsed and incubated with unlabeled glucose, basal 14C0., production was the same in both high K+ and regular Krebs-Ringer bicarbonate buffer. TSH ( 5 mu/ml) significantly increased 14C-lglucose oxidation when the second incubation was done in regular and high K+ Krebs-Ringer bicarbonate buffer. The difference in the basal glucose oxidation in the experiment utilizing a single incubation as compared to a preincubation with lSC-l-glucose reflects several factors. In the latter studies much of the of Preincubation of Dog Thyroid Slices in Krebs-Ringer Bicarbonate Table 3 .-Effect Buffer on Basal and TSH Stimulated 14C-l-Glucose Oxidation * Krebs-Ringer Experimental conditions
Single 45min incubation 1-hr preincubation with 14C-lglucose Single 45min incubation 1-hr preincubation with 14C-lglucose
Bicarbonate
‘“COz-produced (c m/‘a) Buffer Hiah f;+ Krebs-Ringer
Control
TSH (SmU/ml)
43,700?2,800
100,300~4,700
4,400t
200
74,000?4,100
3.900*
300
10,100-c
138,2OO-t9,300
8,300-c
400
82.800 + 2,500
4,400*
200
158.500~10,800
4,800?
Buffer
TSH (SmU/ml)
Control
800
Bicarbonate
200
104,100+- 2.100
9,OOOk
300
173,900+ 13,700
8,300+
400
* The results are the average k SEM of triplicate determinations. Two separate experiments are shown. Slices from the same dog thyroid were used in each experiment. When slices were preincubated with 14C-l-glucose, unlabeled glucose (1 mg/ml) was present during that and the subsequent 45min incubation period. TSH was present only during the second incubation period.
947
EFFECTS OF IONS ON GLUCOSE OXIDATION
14C-l-glucose would have been oxidized to l”COZ and not collected during the first hour of incubation. During the subsequent 45 min of incubation when l*COZ was collected, unlabeled glucose was present in the buffer and this would dilute the specific activity of the precursors of the 14C02 in the cells. Although these results suggests an effect of high K+ to increase glucose transport into the cell, studies utilizing 3H-adenine indicate the effects of K+ are more complicated. Initial incubation of thyroid slices with 3H-adenine and either high K+ or regular Krebs-Ringer bicarbonate buffer, followed by incubation in regular Krebs-Ringer buffer, resulted in formation of similar amounts of 3H-CAMP (Table 4). However, when the second incubation was done using high K+ buffer, 3H-CAMP formation was significantly greater, whether high K+ or regular Krebs-Ringer buffer was used during the first incubation. In general, conversion of 3H-adenine to 3H-CAMP was greater when both the first and second incubations were done with high K+ buffer. In these experiments, effects of TSH and PGEl were very similar to those shown in Tables 1 and 2. In the latter experiments the initial and second incubations were done in the same buffer. Since the high K+ buffer was also low in Na +, the results could reflect changes in either ion. To evaluate more directly the effects of low Na+, choline chloride was substituted for NaCl (Table 5). In some of the experiments the Na+ was totally replaced because KHCO, was used instead of NaHCO.?. Basal glucose oxidation was markedly stimulated with very little additional effect from 3 mu/ml TSH when Na+ was reduced in the buffer. This result was similar to that obtained with high K+ buffer. Incorporation of 3H-adenine into 3H-CAMP and CAMP levels was similar in both buffers, and the response to TSH was very similar. Previously we had reported that exclusion of Ca++ from Krebs-Ringer bicarbonate buffer reduced basal glucose oxidation and the response to a low but not a high dose of TSH. l1 The results in Table 6 indicate that Ca++ is not necessary for TSH stimulation of 3H-adenine conversion to 3H-CAMP or elevation of CAMP concentrations, but is essential for TSH stimulation of glucose oxidation when 3 mu/ml of the hormone is used. In the absence of TSH, exclusion of Ca++ from the buffer did not modify either 3H-CAMP formation or Table 4.-Effect of K+ Concentration of Krebs-Ringer Bicarbonate Buffer on TSH and PGEl Stimulation of 3H-Adenine Incorporation Into 3H-CAMP in Dog Thyroid Slices * Incubation
Buffer
“H-CAMP TSH
First
Second
Regular
Regular
High
High
K+
Regular
High
K+
High
Regular
K+ K+
Control 5.500 20,200
f
(3 mu/ml) 800
(cpm/g) TSH
(50 mU/ml)
PGEl
PGEI
(1 !Jg/mlj
(10 Irg/ml)
17,000
+ 1,200
33,200
f
500
23,600
+
400
27,000
k 4,100
30,200
5
35,500 f
700
29,700
+ 1,300
58.700
+ 2,100
2 5,900
9,000
f
1,400
4,300
5
500
800
* The results are the average & SEM of triplicate determinations. The first incubation was for 1 hr in the appropriate buffer which contained 10 PC sH-adenine and 1 mg/ml glucose. The slices were then rinsed and incubated for 30 min in the appropriate buffer containing 10-s M theophylline and the substance to be tested. The results are representative of six different experiments.
948
YAMASHITA, BLOOM, AND FIELD
Table K-Effect of Absence of Na+ on TSH Stimulation of 14C-l-Glucose Oxidation, CAMP Concentrations, and %Adenine Incorporation into sH-CAMP in Dog Thyroid Slices * Low Na+ Kreb;;Eyr Krebs-Ringer Control
Experiment
Bicarbonate
Bicarbonate
Buffer
TSH (3 mU/ml)
Control
TSH (3 mu/ml)
1
14C0, cpm/g sH-CAMP cpm/g CAMP mm/g Experiment 2 14COs cpm/g sH-CAMP cpm/g CAMP wm/g
39,900 + 2,600 12,500+ 1,100 1.550.2
60,200-t 1,000 22,500a 1,600 3.6kO.3
101,800+ 300 17,300+ 1,200 1.7kO.l
110,400c3,800 32,100? 1,400 3.OkO.2
35,10012,400 10,400t 700 1.220.1
72,6OO-c3,100 30,600” 900 1.820.1
79,600?4,800 10,700-1- 1,100 l.l+-0.1
92,600r5,500 46,600+ 1,300 2.5t0.3
* The results are the average ? SEM of triplicate determinations for glucose oxidation and sH-adenine incorporation into sH-CAMP and quadruplicate determinations for CAMP concentrations. In the low Na+ Krebs-Ringer bicarbonate buffer, the NaCl was replaced with isotonic choline chloride. In Experiment 1, KHCO, was used instead of NaHCO,. In Experiment 2, NaHCO, was used to make the low Nat- Krebs-Ringer bicarbonate buffer.
levels of CAMP. In Ca++-free buffer, 0.1 pg/ml PGE1, as 3 mu/ml TSH, did not increase 14C02 formation (Table 7). Increasing the PGEl to 10 pg/ml, a maximum dose,12 caused stimulation of glucose oxidation, but the effect was less than that produced in buffer containing Ca ++. Despite the decreased effects of PGEl on glucose oxidation, both concentrations of PGEl caused significant increases in 3H-adenine conversion to 3H-CAMP and CAMP concentrations. Although basal values for 3H-CAMP formation and CAMP concentrations were sometimes lower in Ca++-free buffer, the per cent stimulation induced by PGEl was usually the same as in regular Krebs-Ringer bicarbonate buffer. DISCUSSION
The present data confirm our previous results that a high K+ concentration in the buffer mimics the effects of TSH and increases glucose oxidation.12 In those experiments there were no consistent effects of high K+ on CAMP concentration in thyroid slices, and it was postulated that the enhanced 14C02 production reflected changes in glucose transport rather than activation of the adenyl cyclaseCAMP system. Support for an effect of high K+ on membrane transport of glucose was provided by the experiments in which intracellular precursors of 14COn ‘. were labeled prior to incubating thyroid slices in high K+ buffer. Under these conditions (Table 3) basal glucose oxidation was not increased by high K+ buffer, a result which would be anticipated if the primary effect of high K+ were to increase transport of glucose from the medium into the cell. Such an effect of K+ on membrane permeability would be consistent with the observations of Williams who measured membrane potential in thyroid cells.20 He observed that increasing K+ in the buffer was associated with decreasing negativity of the membrane potential. Dekker and Field provided histologic and electron microscopic evidence of morphologic changes in thyroid slices induced by high K+ buffer that would be compatible with alterations in membrane transport.12
Bicarbonate TSH (50 mu/ml)
216,400 e 11,600 14,400 t 2,700 4.9 32.0 +
Buffer
cpm/g
34,400 + 1,200 4,200 t 400 4.9 + 0.7
Control
23,900 t 500 2,300 c 200 1.5 + 0.1
59,700 4 1,000 19,400 & 800 2.1 15.8 t
46,900 + 1,600 11,900 2 1,600 7.6 e 0.8
35,500 f 1,200 8,500 + 500 2.8 -c 0.3
incorporation
into aHCAMP
and quad-
31,000 + 1,000 100 14,000 f 0.5 4.9 2
25,400 t 1,200 4,000 * 100 2.6 f 0.4
Buffer
and
PGEx (10 Irg/mU
Bicarbonate
Concentration
and quad-
PGEi (0.1 wg/ml)
Krebs-Ringer
and
77,200 k 1,000 20,800 k 1,600 5.8 22.7 f into sH-CAMP
CAMP
incorporation
33,900 + 400 8,400 + 600 10.7 +- 2.5
TSH (50 mu/d)
Buffer
AMP Concentration Bicarbonate
TSH (3 mu/ml)
Oxidation, Slices * Cat+-free
Control
Buffer
Control
Bicarbonate
of 14C-l-Glucose in Dog Thyroid
PGEl (10 W/ml)
Krebs-Ringer
of Ca++ From Buffer on PGEl Stimulation 3H-Adenine Incorporation Into 3H-CAMP PGEI (0.1 ~gg/d)
of Exclusion
* The results are the average k SEM of triplicate determinations for glucose oxidation and sH-adenine ruplicate determinations for CAMP concentrations and are representative of three different experiments.
aHCAMP cpm/g CAMP mpm/g
wo,
Table 7.-Effect
Cyclic
Ca++-free Krebs-Ringer
* The results are the average + SEM of triplicate determinations for glucose oxidation and sH-adenine ruplicate determinations for CAMP concentrations and are representative of six different experiments.
76,700 c 1,900 8,300 + 200 8.1 t 1.3
Krebs-Ringer
of Ca+ + From Buffer on TSH Stimulation of ‘4C-l-Glucose Oxidation, 3H-Adeuine Incorporation Into 3H-CAMP in Dog Thyroid Slices *
47,000 +- 1,700 4,200 c 300 4.0 + 0.4
of Exclusion
sH-CAMP cpm/g CAMP mum/g
14cq? cpm/g
Table 6.Effect
950
YAMASHITA,
BLOOM, AND FIELD
However, the role of K+ is certainly more complex than simply altering glucose transport into the cell. Previously we had demonstrated that insulin augmented glucose uptake by thyroid slices but did not stimulate glucose oxidation.?’ The data in Table 4 do not indicate an effect of high K+ on 3H-adenine uptake into the cell. Basal 3H-CAMP formation was essentially the same when high K+ and regular Krebs-Ringer bicarbonate buffers were used during the incubation with 3H-adenine and a regular buffer used for the subsequent incubation. Examination of the results obtained when slices were incubated in regular Krebs-Ringer bicarbonate buffer and 3H-adenine and then transferred to either regular or high K+ buffer clearly indicates that K+ in the second incubation augmented 3H-CAMP formation. This could reflect either an effect of K+ on “H-ATP formation from 3H-adenine or increased :$H-ATP conversion to “HCAMP. The absence of a clear-cut stimulation of CAMP concentrations by high K+l” militates against the second possibility. This is different from the results reported in other tissues. High K+ in the buffer increased CAMP concentrations in parotid gland,“” brain,“” and rat diaphragm.2J The apparent higher specific activity of the CAMP in the presence of K+ would be compatible with increased incorporation of “H-adenine into 3H-ATP. In these experiments, the high K+ buffer was also deficient in Na+, raising the possibility that the results reflected absence of this iron rather than surfeit of K+. The results in Table 5 indicate that the reduction in Na+ is probably more important than the excess of K+ since replacement of NaCl with choline chloride also increased glucose oxidation but decreased TSH stimulation of l:‘CO,. The effect of TSH on 3H-adenine conversion to 3H-CAMP and CAMP concentrations was similar in buffer with normal and reduced amounts of Na+. This is again consistent with the concept that alterations in ionic composition of the buffer modify effects of CAMP and not its generation. The role of Ca++ in mediating the effects of TSH on various aspects of thyroid function has also been examined. Zor et al. reported that basal glucose oxidation was decreased in thyroid slices incubated in the absence of Ca++ and that the TSH (3 mu/ml) stimulation of W02 production and 32P incorporation into phospholipid was inhibited .ll However, increasing the concentration of TSH to 50 mu/ml appeared to overcome the inhibition. Since the effects of the dibutyryl derivative of CAMP were also reduced in the absence of Ca++, it was suggested that Ca++ was essential for the action of the cyclic nucleotide rather than for its formation. Dekker and Field provided additional support for this conclusion since elimination of Ca ++ from the buffer did not diminish the TSH increase in CAMP.l” The dose of TSH was ineffective in stimulating glucose oxidation in the absence of Ca ++. Previously Bakke et al. had reported that Ca++ was essential for the action of TSH on increasing thyroid slice weight.“’ Ca++ also appeared to be necessary for incorporating Is11 into thyroglobulin.2G In contrast to these effects, TSH was still capable of stimulating colloid droplet formation in the absence of Ca ++.I2 Thus, there is a dissociation between Ca++ dependence and the various actions of CAMP. The data in Table 6 confirm the results that Ca++ is not essential for TSH stimulation of the adenyl cyclase-CAMP system. Both the low and the high doses of TSH caused equivalent increments in CAMP levels and 3H-adenine conversion
EFFECTS
OF IONS ON GLUCOSE OXIDATION
951
to 3H-CAMP whether Ca++ was present in the buffer or not. These results are somewhat at variance with those obtained when the adenyl cyclase activity of homogenates has been measured. Burke reported that Ca++ decreased basal adenyl cyclase activity and inhibited the stimulation induced by TSH and F- in homogenates of sheep thyroid glands. 27 Bar and Hechter observed that EGTA increased basal adenyl cyclase activity in mitochondrial and microsomal fractions of bovine adrenal cortex and rat fat cell ghosts but abolished stimulation caused by ACTH.28 Bradham et al. found that EGTA inhibition of adenyl cyclase activity of homogenates of calf cerebral cortext could be reversed by addition of Ca++.3” In contrast to our results, Kuo noted that TSH stimulation of 3H-adenine incorporation into 3H-CAMP in isolated adipose cells was dependent upon Ca++.30 In his experiments Ca++ was also essential for the action of ACTH and LH but not for norepinephrine and glucagon. Such differences were interpreted as reflecting differences in the binding of the hormones to the cell receptor and their subsequent activation of adenyl cyclase. There is no compelling reason why the receptor sites of one tissue might require Ca++ while those of another might not even though the same hormone was involved in the stimulation. Certainly the effects of TSH in thyroid appear to be much more specific than on adipose tissue. Experiments utilizing intact cells in tissue slices are probably more meaningful than those measuring effects of ions on adenyl cyclase in tissue homogenates. An excellent example of this is the dissociation of the effects of Fon the adenyl cyclase-CAMP system in thyroid homogenate and intact cells in slicesI The mechanism by which Ca++ is essential for the expression of effects of CAMP is not known. This problem as well as the relationship between Ca++ and hormone action was recently reviewed by Rasmussen.3r Studies were done using PGE, and TSH in hopes that additional information would be obtained related to the mechanism of their action on various parameters of thyroid function. Although PGEl mimicked many of the effects of TSH on the adenyl cyclase-CAMP system, on glucose oxidation, and 32P incorporation into phospholipids, some of the results suggested that its action might be mediated by a mechanism different from that of TSH.32 In general, the effects of PGE, in high K+ buffer were similar to those obtained using TSH. Although PGE, caused greater stimulation of CAMP levels in slices incubated in high K+ buffer (Table 2), this was not a consistent finding and did not indicate greater responsiveness of adenyl cyclase activity to PGE, under these circumstances. These results, and those with TSH, indicate that high K+ interfered with the action of CAMP and not its generation induced by PGE,. The results obtained with PGEl in Ca ++-free buffer were also qualitatively similar to those observed with TSH. The low dose of PGEl (0.1 Irg/ml) was completely ineffective in increasing glucose oxidation while a higher dose (10 pg/ml) caused a significant effect. Although 0.1 pg/ml PGEl did not increase 14C02 formation, it still stimulated 3H-adenine incorporation into 3H-CAMP and CAMP concentration, further supporting the role of Ca++ in the expression of the effects of CAMP rather than its generation. The apparent reduction of PGE, stimulation of adenyl cyclase-CAMP system in high K+ buffer (Table 7) was not consistently found in all experiments. The absence of clear-cut differ-
952
YAMASHITA, BLOOM, AND FIELD
ences between the responses to TSH and PGEl in high K+ or Ca++-fkee buffers does not necessarily indicate that they produce these changes by the same mechanism or that they stimulate adenyl cyclase activity by the same receptors. ACKNOWLEDGMENT We are indebted to Mrs. Loretta Malley and Miss Barbara Sheehan for expert secretarial assistance. Bovine TSH (2 U/mg) was a generous gift from the Endocrinology Study Section, National Institutes of Health. Prostaglandin E, was kindly provided by Dr. John Pike of the Upjohn Company, Kalamazoo, Mich. 14C-l-glucose (2.4 mCi/mmole) was purchased from Amersham-Searle Corporation. sH-adenine (22 Ci/mmole) and aH-CAMP (2.35 Ci/mmole) were obtained from Schwartz Bio-Research Corporation. REFERENCES 1. Douglas, W. W.: A possible mechanism of neurosecretion: Release of vasopressin by depolarization and its dependence on calcium. Nature 197:81, 1963. 2. -, and Rubin, R. P.: The role of calcium in the secretory response of the adrenal medulla to acetylcholine. J. Physiol. 159:40, 1961. 3. Samli, M. H., and Geschwind, I. I.: Some effects of energy-transfer inhibitors and of Ca++-free or K+-enhanced media on the release of luteinizing hormone (LH) from the rat pituitary gland in vitro. Endocrinology 82:225, 1968. 4. Kraicer, J., Milligan, J. V., Gosbee, J. L., Conrad, R. G., and Branson, C. M.: In vitro release of ACTH: Effects of potassium, calcium and corticosterone. Endocrinology 85: 1144, 1969. 5. Lambert, A. E., Jeanrenaud, B., Junod, A., and Renold, A. E.: Organ culture of fetal rat pancreas. II. Insulin release induced by amino and organic acids, by hormonal peptides, by cationic alterations of the medium and by other agents. Biochim. Biophys. Acta 184:540, 1969. 6. Birmingham, M. K., Elliott, F. H., and Valere, P. H.-L.: The need for the presence of calcium for the stimulation in vitro of rat adrenal glands by adrenocorticotrophic hormone. Endocrinology 53:687, 1953. 7. Vale, W., Burgus, R., and Guillemin, R.: Presence of calcium ions as a requisite for the in vitro stimulation of TSH-release by hypothalamic TRF. Experientia 23:853, 1967. 8. Parsons, J. A.: Calcium ion requirement for prolactin secretion by rat adenohypophyses in vitro. Amer. J. Physiol. 217: 1599, 1969. 9. Oka, H., and Field, J. B.: Effects of ions on TSH stimulation of s*P incorpora-
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