Pergamon Preea
Life S iences Vol . 9, pp . 35-40, 1976 Printe in the U .S .~ .
EFFECTS OF 8 BROMO-CYCLIC GMP ON CYCLIC AMP LEVELS IN URINE AND TISSUES OF HYPOTHYROID RATS J .A . Fernandez-Pol* and M .T . Hays Nuclear Medicine Service, Veterans Administration Hospital, Buffalo, New York
14215 and the Departments of Medicine,
Nuclear Medicine and Biophysical Sciences of the State University of New York at Buffalo, Buffalo, New York
14214
(Received in final form May 17, 1976) SUMMARY The effects of 8-bromo-guanosine 3',5' cyclic monophosphate (8Brcyclic GMP) on the levels of cyclic AMP in urine, brain, liver, and muscle were determined in hypothyroid rats . Cyclic AMP urinary levels were significantly lower in untreated hypothyroid rats than in normal rats, and when hypothyroid rats were treated with replacement thyroxine, urinary cyclic AMP returned to normal after six days of treatment . Hypothyroid rats treated with 8Brcyclic GMP, 30 mg/kg body weight subcutaneously every 12 hours exhibited a biphasic response . From days 2 through 4 of treatment, cyclic AMP excretion rose to approximately 2 .5 times normal levels . Subsequently, the cyclic AMP excretion returned to hypothyroid levels and remained low throughout the rest of the treatment period . Tissue cyclic AMP levels in hypthyroid rats treated with 8Br-cyclic GMP were increased in comparison to those of untreated hypothyroid rats . Increase in brain was 134%, in liver 55%, and in muscle 42% . We conclude from these studies that 8Br-cyclic GMP can significantly alter cyclic AMP levels in hypothyroid rats . It was first shown by Hardman et . a l . that thyroparathyroidectomized rats have decreased urinary levels of both cyclic GMP and cyclic AMPS, and that these low levels were restored to normal or near normal levels by maintenance doses of thyroxine (1) . Others have shown that in humans and in dogs, thyroid
* Present address : Laboratory of Molecular Biology, National Cancer Institute, NIH, Bethesda, Maryland 20014 . ~ Abbreviations used in this paper : cyclic AMP : cyclic 3' :5', Adenosine monophosphate ; cyclic GMP : cyclic 3' :5', Guanosine monophosphate ; 8Br cyclic GMP : 8-Br~omo-cyclic 3' :5' Guanosine Monophosphate ; T3 : triiodothyronine ; T4 : tetraiiodothyronine (Thyroxine) .
35
36
8Br-Ggclic GMP _and Hypothyroidism
Vol . 19, No . 1
hormones can affect the levels of cyclic GMP and cyclic AMP in vivo (2, 3, 4) . Evidence also exists that exogenous cyclic GMP elicits cyclic AMP-like effects and that these effects might be attributed in some cases to alterations in cyclic AMP metabolism (5) . These findings show on the one hand a relation between thyroid hormones and cyclic nucleotides and on the other, between cyclic P.MP and cyclic GMP (5) . The purpose of the present investigation was to study the effects of 8Br-cyclic GMP on cyclic AMP levels in hypothyroid rats . Materials and Methods Materials : Thyroxine was purchased from Flint Laboratories ; 8Br-cyclic GMP r~gma (St . Louis, Miss .) and 8- 3H adenosine 3',5' cyclic monophosphate in 50% ethanol from Schwarz/Mann (for recovery studies) ; Cyclic AMP radioimmunoassays kits from Schwarz/Mann . The experimental substances were sterilized by Millipore filtration and injected in a 0 .5 ml volume . ~P_re__paration of Animals : Male Sprague-Dawley rats (purchased from Charles River La~orator es, Mass . , weighing approximately 40-50 g . at the beginning of the study were utilized in these experiments . Thyroidectomy was performed at age 21 days . A few rats from whom the parathyroid glands had inadvertently been removed developed tetany and died within 2-3 days . Other animals exhibiting irritability were not used . All animals were maintained on Purina Lab Chow and water at lib (1% calciian gluconate was added to water since the possibility of parathyroid alteration was not ruled out until the end of the study) . Completeness of thyroidectomy was checked by autopsy . All animals were housed in metabolic cages (two to three rats in a single metabolic cage) . Number of rats in each group was 9 to 12 . 1 . H oth roid rou Ex erimental Desi n : Rats were studied in four groups : Hypot yroid, t yroidectomized, saline injected . 2 . 8Br-c clic P rou ays w t BrThyroidectomized at 21 days of age and treated from age cyclic GMP dissolved in 8% gelatin, 30 mg/kg/BW, subcutaneously every 12 hours . 3 . T4 rou : Thyroidectomized at 21 days of age and treated from age 42-52 days wit T4, 2-3 ,~e g/day (T4 was . dissolved in normal saline solution) . 4 . Control : Unoperated, saline injected . Rats in all groups were killed after 10 days of treatment . Observations : Growth Curve (FIG . 1) : The growth curves were used in order to assess the thyroid status 6) and to determine the effects of 8Br-cyclic GMP and T4 upon body weight . All rats were weighed every 24 hours . The injection of the experimental substances started when the difference in weight of the control in comparison to the experimental groups was significantly different (level of significance P < 0 .05), thus, demonstrating a hypothyroid state (6) . Urine : Urine samples consisted of the 24-hour excretion of groups of 2 or 3 rats housed in a single metabolic cage . Urine was collected as described by Hardman et . al . (1) . Tissue : Rats were anesthetized with 35 mg/kg/BW of sodium pentobarbital I .P . ~parotort~y was performed with exposure of the liver . A thigh muscle was also exposed and portions of liver and skeletal muscle were frozen in situ between stainless steel tongs precooled in liquid nitrogen . Then, they were cut by scalpel and plunged in liquid nitrogen . Brain tissue was obtained as described by Steiner (7) . Samples were processed as described by Steiner (8) . C clic AMP was measured by radioimmunoassay after prior purification of samples y Dowex Columns (3) . The doses of 8Br-cyclic GMP used did not interfere with cyclic AMP measurement by radioimmunoassay (unpublished observations) . Basal adenyl cyclase was measured by the method of Khrisna (g) . Tissu~roteins were determined by the method of Lowry (10) .
Vol . 19, No . 1
37
BBrG~+clic (~ and Hypothyroidism
FIG . 1 Effects of 8Br-cyclic GMP and T4 treatment on body weight . "+r"wn : Control ; " . """"""" . : Hypothyroid ; ~ " ~ .~ : Hypothyroid T4 treated ; ""~ir~~u~ : Hypothyroid 8Br-cyclic GMP treated . P : Probability value (see Methods) . Anal sis of Radioimmunoassa Data : The radioimmunoassay data was analyzed us ng a computer program eve oped by Rodbard and Lewald, with minor modifications introduced in this laboratory (3) . The interassay coefficient of variation was 12% and the intraassay coefficient of variation 8% . All samples were measured in duplicate . Statistical analyses were performed using the StudentFisher test (11) . Results Growth Curve (FIG . 1) : Hypothyroid rats treated with T4 showed a steady ncre~ase body weight and the slope of the growth curve becart~e normal after 5-6 days of treatment . 8Br-cyclic GMP did not produce any effect on body weight of hypothyroid animals .
in
Urine (FIG . 2) : 1. oth raid rou : This group had lower cyclic AMP levels -tfincontrol (P < 0 .0 . .~8Br-c cl_i~ c GMP rou~: Hypothyroid rats treated with 8Br-cyclic GMP had a peak in cyclic AF'IP urinary excretion from day 2 through 4 of treatment, rising 2 .5 times above control levels . Subsequently, cyclic AMP excretion returned to hypothyroid levels despite continuous treatment . 3 . Tq group : In hypothyroid rats treated with Tq, cyclic AMP excretion became normal after 6 days of treatment . 4 . Control : The cyclic AMP levels did not change significantly throughout the stuffy. Tissues (FIGS . 3, 4, and 5) : a . In all tissues studied cyclic AMP levels were nc~ reared in rats treated with 8Br-cyclic GMP (P < 0 .01) . Increase in brain was 134%, in liver 55%, and in muscle 42% in comparison to control ; b . In rats treated with T , cyclic AMP was found to be significantly increased liver only (FIG . 3) ~P < 0 .05) ; _c . Adenyl cyclase did not change significantly in any of the groups studied .
in
Discussion Several studies have appeared suggesting either a direct or an indirect effect of thyroid status upon cyclic nucleotide metabolism (3) . Alterations
38
Vol . 19, No . 1
8Br-Cyclic GMP and Hypothyroidism
~60 140
I
2
3
4
5
6
7
8
9
10
FIG . 2
Effects of 8Br-cyclic GMP and Tq treatment on urinary cyclic AMP levels . are as in FIG . 1 .
Symbols
FIG . 3
Effects of 8Br-cyclic GMP and Tq treatment on cyclic AMP levels .
in adenyl cyclase, phosphodiesterase, cyclic AMP and cyclic GMP levels have been observed under altered thyroid function (3) . Interactions between cyclic AMP and cyclic GMP have been extensively documented (5) . In our studies, 8Br-cyclic GMP was used because of its greater resistence to hydrolysis as well as its easy penetrability into cells in comparison to cyclic GMP (12) . We found that cyclic GMP in a variety of doses had no effect on cyclic AMP levels in either control or hypothyroid animals (unpublished observations) .
Vol . 19, No . 1
8Br-Cyclic G?1P and 8ypothyroidiam
Effects of 8Br-cyclic GMP and T4 treatment on cyclic AMP levels .
39
Effects of 8Br-cyclic GMP and T4 treatment on cyclic AMP levels .
A comparison group of hypothyroid animals was treated with T4 in order to investigate the effects of T4 on cyclic AMP levels and to compare its effects with those of 8Br-cyclic GMP . In urine, hypothyroid rats (FIG . 2) had lower cyclic AMP levels than control . These findings confirm the observation of Hardman et . al . (1) on the effect of thyroid hormones upon urinary cyclic nucleotides . Replacement therapy with T4 produced, after a lag period of 6 days, .a significant increase in urinary cyclic AMP levels (FIG . 2) . The urinary cyclic AMP levels in the 8Br-cyclic GMP treated group showed increased cyclic AMP levels for 72 hours (FIG . 2) . These early cyclic AMP levels were higher than normal . Subsequently, despite continued treatment, the levels became hypothyroid again . The increase in urinary cyclic AMP levels might be either a direct effect of 8Br-cyclic GMP upon kidney (e .g . : increase in cyclic AMP excretion produced by inhibition of kidney phosphodiesterase(s)), or, since these studies were carried out in vivo, it is possible that the effects on urinary cyclic AMP were indirect~econdary to effects of 8Br-cyclic GMP at other sites or tissues (e .g . : release of hormones that in turn would increase urinary cyclic AMP) . These effects of 8Br-cyclic GMP appear characteristic of the hypothyroid animal . In pilot studies with doses up to five-fold those reported here, 8Br-cyclic GMP had no effect on tissue and urine cyclic AMP levels in enthyroid rats . From the tissues studied, only in liver have we detected significantly higher cyclic AMP levels than control in hypothyroid rats treated with T4 (FIG . 3) . This increase in liver cyclic AMP levels might be due to an inhibition of phosphodiesterase(s) by T4 after conversion to T3 (13) . Indeed, T3 is the only hormone which has been shown to inhibit phosphodiesterase (14) . This finding points out that the liver might be one of the sources of increased urinary cyclic AMP in the T4 treated group . Further studies are necessary to explore these points . 8Br-cyclic GMP treatment produced an increase in cyclic AMP levels in all tissues studied (FIGS . 3, 4, and 5) . In our studies we could not show any significant difference in basal adenylate cyclase activity in any of the groups studied . This finding is in agreement with the measurements of J .K . Jones
40
8Br-Cyclic GMP and Hypothyroidism
Vol . 19, No . 1
et . al . (15) . We would attribute the elevated tissue cyclic AMP levels to an inhibition of phosphodiesterase activity by 8Br-cyclic GMP, with the increased cyclic AMP levels in tissues due to prevention of the hydrolysis of cyclic AMP . Direct measurement of phosphodiesterase activity is needed to confirm this assumption . However, Van Inwegen et . a l . have shown that phosphodiesterase activity is altered in hypothyroid rats (16) . Our results suggest that phosphodiesterase might be more easily inhibited by 8Br-cyclic GMP in hypothyroid rats . The present experiments must be interpreted as preliminary and within the framework of the limitations emphasized above . However, they show that 8Brcyclic GMP is capable of altering concentrations of cyclic AMP levels in urine and tissues of hypothyroid rats, and that the pharmacological effects of 8Brcyclic (~iP might be mediated by a nonspecific increase in cyclic AMP levels in tissues . Acknowled ements : This work was supQorted by Veterans Administration Advanced pec a ty ra n ng Program - TR-#158 and by Veterans Administration designated research funds . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 .
J .G . HARDMAN, J .W . DAVIS and E .W . SUTHERLAND, J . Biol . Chem . 244, 63546362 (1969) . T . LIN, L .E . KOPP and J .R . TUCCI . J . Clin . Endocrinol . Metab . 36, 10331036 (1973) . J .A . FERNANDEZ-POL and M .T . HAYS . Acta Endocrinol . (Kovenhavn) , 79, 66-75 (1975) . B .E . KARLBERG, K .G . HENRIKSSON and R .G . ANDERSSON . J . Clin . Endocrinol . Metab . 39, 96-101 (1974) . In Advances in C clic ~~OLDBERG, R .F . O'DEA and M .K, HADDOX . Nucleotide Research, Yol . 3, p . 155-223 . P . Greengar and G . . Robison, E hors, aven ress, N .Y . (1973) . E .B . ASTWOOD . In the Pharmacolo ical Basis of Thera eutics . 4th Edition, p . 1473 . L .S . Goodma n and . Gilman, Editors . T e Maani an Co . (1970) . A .L . STEINER, J .A . FERRENDELLI and D .M . KIPNIS . J . Biôl . Chem . 247, 1121-1124 (1972) . A .L . STEINER, R .E . WEHMANN, C .W . PARKER and D .M . KIPNIS . In Advances in P, Greengard, G .A . Robison C clic Nucleotide Research, Vol . 2, p . 51-61 . an . ao ett , tors, Raven Press, N,Y . (1972) . G . KRISHNA, B . WEISS and B .B . BRODIE . J, of Pharmacology and Experimental Therapeutics , 163, 379-385 (1968) . O .H . LOWRY, M .J . ROSEBROUGH, A .L . FARR and R .J . RANDAL . J . Biol . Chem . 193, 265-270 (1951) . G .W . SNEDECOR and W .G . COCHRAN . Statistical Methods, 6th Edition . The 36 Iowa State University Press, Ames In Advances in C clic Nucleotide L .N . SIMON, D .A . SHUMAN and R,K . ROBINS . ors, Research , Vol . 3, p . 225-353 . P . Greengard an G . . Ro son, Raven Press, N .Y . (1973) . 91, 934-947 (1972) . S . REFETOFF, R . MATALON and M . BIGAZZI . Endocrinolo G .A . ROBISON, R .W . BUTCHER and E .W . SUTHERL yc is AMP , 1st Edition, p . 28, Academic Press, N .Y . and London (1971) . J .K . JONES, F . ISPiAIL-BEIGI and I .S . EDELMAN . J . Clin, Invest . 51, 24982501 (1972) . R .G . VAN INWEGEN, G . ALAN ROBISON, W .J . THOMPSON, K .J . ARMSTRONG and J .E . STOUFFER . J . Biol . Chem . 250, 2452-2456 (1975) .
Life S iences Vol . 9, pp . 35-40, 1976 Printe in the U .S .~ .
EFFECTS OF 8 BROMO-CYCLIC GMP ON CYCLIC AMP LEVELS IN URINE AND TISSUES OF HYPOTHYROID RATS J .A . Fernandez-Pol* and M .T . Hays Nuclear Medicine Service, Veterans Administration Hospital, Buffalo, New York
14215 and the Departments of Medicine,
Nuclear Medicine and Biophysical Sciences of the State University of New York at Buffalo, Buffalo, New York
14214
(Received in final form May 17, 1976) SUMMARY The effects of 8-bromo-guanosine 3',5' cyclic monophosphate (8Brcyclic GMP) on the levels of cyclic AMP in urine, brain, liver, and muscle were determined in hypothyroid rats . Cyclic AMP urinary levels were significantly lower in untreated hypothyroid rats than in normal rats, and when hypothyroid rats were treated with replacement thyroxine, urinary cyclic AMP returned to normal after six days of treatment . Hypothyroid rats treated with 8Brcyclic GMP, 30 mg/kg body weight subcutaneously every 12 hours exhibited a biphasic response . From days 2 through 4 of treatment, cyclic AMP excretion rose to approximately 2 .5 times normal levels . Subsequently, the cyclic AMP excretion returned to hypothyroid levels and remained low throughout the rest of the treatment period . Tissue cyclic AMP levels in hypthyroid rats treated with 8Br-cyclic GMP were increased in comparison to those of untreated hypothyroid rats . Increase in brain was 134%, in liver 55%, and in muscle 42% . We conclude from these studies that 8Br-cyclic GMP can significantly alter cyclic AMP levels in hypothyroid rats . It was first shown by Hardman et . a l . that thyroparathyroidectomized rats have decreased urinary levels of both cyclic GMP and cyclic AMPS, and that these low levels were restored to normal or near normal levels by maintenance doses of thyroxine (1) . Others have shown that in humans and in dogs, thyroid
* Present address : Laboratory of Molecular Biology, National Cancer Institute, NIH, Bethesda, Maryland 20014 . ~ Abbreviations used in this paper : cyclic AMP : cyclic 3' :5', Adenosine monophosphate ; cyclic GMP : cyclic 3' :5', Guanosine monophosphate ; 8Br cyclic GMP : 8-Br~omo-cyclic 3' :5' Guanosine Monophosphate ; T3 : triiodothyronine ; T4 : tetraiiodothyronine (Thyroxine) .
35
36
8Br-Ggclic GMP _and Hypothyroidism
Vol . 19, No . 1
hormones can affect the levels of cyclic GMP and cyclic AMP in vivo (2, 3, 4) . Evidence also exists that exogenous cyclic GMP elicits cyclic AMP-like effects and that these effects might be attributed in some cases to alterations in cyclic AMP metabolism (5) . These findings show on the one hand a relation between thyroid hormones and cyclic nucleotides and on the other, between cyclic P.MP and cyclic GMP (5) . The purpose of the present investigation was to study the effects of 8Br-cyclic GMP on cyclic AMP levels in hypothyroid rats . Materials and Methods Materials : Thyroxine was purchased from Flint Laboratories ; 8Br-cyclic GMP r~gma (St . Louis, Miss .) and 8- 3H adenosine 3',5' cyclic monophosphate in 50% ethanol from Schwarz/Mann (for recovery studies) ; Cyclic AMP radioimmunoassays kits from Schwarz/Mann . The experimental substances were sterilized by Millipore filtration and injected in a 0 .5 ml volume . ~P_re__paration of Animals : Male Sprague-Dawley rats (purchased from Charles River La~orator es, Mass . , weighing approximately 40-50 g . at the beginning of the study were utilized in these experiments . Thyroidectomy was performed at age 21 days . A few rats from whom the parathyroid glands had inadvertently been removed developed tetany and died within 2-3 days . Other animals exhibiting irritability were not used . All animals were maintained on Purina Lab Chow and water at lib (1% calciian gluconate was added to water since the possibility of parathyroid alteration was not ruled out until the end of the study) . Completeness of thyroidectomy was checked by autopsy . All animals were housed in metabolic cages (two to three rats in a single metabolic cage) . Number of rats in each group was 9 to 12 . 1 . H oth roid rou Ex erimental Desi n : Rats were studied in four groups : Hypot yroid, t yroidectomized, saline injected . 2 . 8Br-c clic P rou ays w t BrThyroidectomized at 21 days of age and treated from age cyclic GMP dissolved in 8% gelatin, 30 mg/kg/BW, subcutaneously every 12 hours . 3 . T4 rou : Thyroidectomized at 21 days of age and treated from age 42-52 days wit T4, 2-3 ,~e g/day (T4 was . dissolved in normal saline solution) . 4 . Control : Unoperated, saline injected . Rats in all groups were killed after 10 days of treatment . Observations : Growth Curve (FIG . 1) : The growth curves were used in order to assess the thyroid status 6) and to determine the effects of 8Br-cyclic GMP and T4 upon body weight . All rats were weighed every 24 hours . The injection of the experimental substances started when the difference in weight of the control in comparison to the experimental groups was significantly different (level of significance P < 0 .05), thus, demonstrating a hypothyroid state (6) . Urine : Urine samples consisted of the 24-hour excretion of groups of 2 or 3 rats housed in a single metabolic cage . Urine was collected as described by Hardman et . al . (1) . Tissue : Rats were anesthetized with 35 mg/kg/BW of sodium pentobarbital I .P . ~parotort~y was performed with exposure of the liver . A thigh muscle was also exposed and portions of liver and skeletal muscle were frozen in situ between stainless steel tongs precooled in liquid nitrogen . Then, they were cut by scalpel and plunged in liquid nitrogen . Brain tissue was obtained as described by Steiner (7) . Samples were processed as described by Steiner (8) . C clic AMP was measured by radioimmunoassay after prior purification of samples y Dowex Columns (3) . The doses of 8Br-cyclic GMP used did not interfere with cyclic AMP measurement by radioimmunoassay (unpublished observations) . Basal adenyl cyclase was measured by the method of Khrisna (g) . Tissu~roteins were determined by the method of Lowry (10) .
Vol . 19, No . 1
37
BBrG~+clic (~ and Hypothyroidism
FIG . 1 Effects of 8Br-cyclic GMP and T4 treatment on body weight . "+r"wn : Control ; " . """"""" . : Hypothyroid ; ~ " ~ .~ : Hypothyroid T4 treated ; ""~ir~~u~ : Hypothyroid 8Br-cyclic GMP treated . P : Probability value (see Methods) . Anal sis of Radioimmunoassa Data : The radioimmunoassay data was analyzed us ng a computer program eve oped by Rodbard and Lewald, with minor modifications introduced in this laboratory (3) . The interassay coefficient of variation was 12% and the intraassay coefficient of variation 8% . All samples were measured in duplicate . Statistical analyses were performed using the StudentFisher test (11) . Results Growth Curve (FIG . 1) : Hypothyroid rats treated with T4 showed a steady ncre~ase body weight and the slope of the growth curve becart~e normal after 5-6 days of treatment . 8Br-cyclic GMP did not produce any effect on body weight of hypothyroid animals .
in
Urine (FIG . 2) : 1. oth raid rou : This group had lower cyclic AMP levels -tfincontrol (P < 0 .0 . .~8Br-c cl_i~ c GMP rou~: Hypothyroid rats treated with 8Br-cyclic GMP had a peak in cyclic AF'IP urinary excretion from day 2 through 4 of treatment, rising 2 .5 times above control levels . Subsequently, cyclic AMP excretion returned to hypothyroid levels despite continuous treatment . 3 . Tq group : In hypothyroid rats treated with Tq, cyclic AMP excretion became normal after 6 days of treatment . 4 . Control : The cyclic AMP levels did not change significantly throughout the stuffy. Tissues (FIGS . 3, 4, and 5) : a . In all tissues studied cyclic AMP levels were nc~ reared in rats treated with 8Br-cyclic GMP (P < 0 .01) . Increase in brain was 134%, in liver 55%, and in muscle 42% in comparison to control ; b . In rats treated with T , cyclic AMP was found to be significantly increased liver only (FIG . 3) ~P < 0 .05) ; _c . Adenyl cyclase did not change significantly in any of the groups studied .
in
Discussion Several studies have appeared suggesting either a direct or an indirect effect of thyroid status upon cyclic nucleotide metabolism (3) . Alterations
38
Vol . 19, No . 1
8Br-Cyclic GMP and Hypothyroidism
~60 140
I
2
3
4
5
6
7
8
9
10
FIG . 2
Effects of 8Br-cyclic GMP and Tq treatment on urinary cyclic AMP levels . are as in FIG . 1 .
Symbols
FIG . 3
Effects of 8Br-cyclic GMP and Tq treatment on cyclic AMP levels .
in adenyl cyclase, phosphodiesterase, cyclic AMP and cyclic GMP levels have been observed under altered thyroid function (3) . Interactions between cyclic AMP and cyclic GMP have been extensively documented (5) . In our studies, 8Br-cyclic GMP was used because of its greater resistence to hydrolysis as well as its easy penetrability into cells in comparison to cyclic GMP (12) . We found that cyclic GMP in a variety of doses had no effect on cyclic AMP levels in either control or hypothyroid animals (unpublished observations) .
Vol . 19, No . 1
8Br-Cyclic G?1P and 8ypothyroidiam
Effects of 8Br-cyclic GMP and T4 treatment on cyclic AMP levels .
39
Effects of 8Br-cyclic GMP and T4 treatment on cyclic AMP levels .
A comparison group of hypothyroid animals was treated with T4 in order to investigate the effects of T4 on cyclic AMP levels and to compare its effects with those of 8Br-cyclic GMP . In urine, hypothyroid rats (FIG . 2) had lower cyclic AMP levels than control . These findings confirm the observation of Hardman et . al . (1) on the effect of thyroid hormones upon urinary cyclic nucleotides . Replacement therapy with T4 produced, after a lag period of 6 days, .a significant increase in urinary cyclic AMP levels (FIG . 2) . The urinary cyclic AMP levels in the 8Br-cyclic GMP treated group showed increased cyclic AMP levels for 72 hours (FIG . 2) . These early cyclic AMP levels were higher than normal . Subsequently, despite continued treatment, the levels became hypothyroid again . The increase in urinary cyclic AMP levels might be either a direct effect of 8Br-cyclic GMP upon kidney (e .g . : increase in cyclic AMP excretion produced by inhibition of kidney phosphodiesterase(s)), or, since these studies were carried out in vivo, it is possible that the effects on urinary cyclic AMP were indirect~econdary to effects of 8Br-cyclic GMP at other sites or tissues (e .g . : release of hormones that in turn would increase urinary cyclic AMP) . These effects of 8Br-cyclic GMP appear characteristic of the hypothyroid animal . In pilot studies with doses up to five-fold those reported here, 8Br-cyclic GMP had no effect on tissue and urine cyclic AMP levels in enthyroid rats . From the tissues studied, only in liver have we detected significantly higher cyclic AMP levels than control in hypothyroid rats treated with T4 (FIG . 3) . This increase in liver cyclic AMP levels might be due to an inhibition of phosphodiesterase(s) by T4 after conversion to T3 (13) . Indeed, T3 is the only hormone which has been shown to inhibit phosphodiesterase (14) . This finding points out that the liver might be one of the sources of increased urinary cyclic AMP in the T4 treated group . Further studies are necessary to explore these points . 8Br-cyclic GMP treatment produced an increase in cyclic AMP levels in all tissues studied (FIGS . 3, 4, and 5) . In our studies we could not show any significant difference in basal adenylate cyclase activity in any of the groups studied . This finding is in agreement with the measurements of J .K . Jones
40
8Br-Cyclic GMP and Hypothyroidism
Vol . 19, No . 1
et . al . (15) . We would attribute the elevated tissue cyclic AMP levels to an inhibition of phosphodiesterase activity by 8Br-cyclic GMP, with the increased cyclic AMP levels in tissues due to prevention of the hydrolysis of cyclic AMP . Direct measurement of phosphodiesterase activity is needed to confirm this assumption . However, Van Inwegen et . a l . have shown that phosphodiesterase activity is altered in hypothyroid rats (16) . Our results suggest that phosphodiesterase might be more easily inhibited by 8Br-cyclic GMP in hypothyroid rats . The present experiments must be interpreted as preliminary and within the framework of the limitations emphasized above . However, they show that 8Brcyclic GMP is capable of altering concentrations of cyclic AMP levels in urine and tissues of hypothyroid rats, and that the pharmacological effects of 8Brcyclic (~iP might be mediated by a nonspecific increase in cyclic AMP levels in tissues . Acknowled ements : This work was supQorted by Veterans Administration Advanced pec a ty ra n ng Program - TR-#158 and by Veterans Administration designated research funds . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 .
J .G . HARDMAN, J .W . DAVIS and E .W . SUTHERLAND, J . Biol . Chem . 244, 63546362 (1969) . T . LIN, L .E . KOPP and J .R . TUCCI . J . Clin . Endocrinol . Metab . 36, 10331036 (1973) . J .A . FERNANDEZ-POL and M .T . HAYS . Acta Endocrinol . (Kovenhavn) , 79, 66-75 (1975) . B .E . KARLBERG, K .G . HENRIKSSON and R .G . ANDERSSON . J . Clin . Endocrinol . Metab . 39, 96-101 (1974) . In Advances in C clic ~~OLDBERG, R .F . O'DEA and M .K, HADDOX . Nucleotide Research, Yol . 3, p . 155-223 . P . Greengar and G . . Robison, E hors, aven ress, N .Y . (1973) . E .B . ASTWOOD . In the Pharmacolo ical Basis of Thera eutics . 4th Edition, p . 1473 . L .S . Goodma n and . Gilman, Editors . T e Maani an Co . (1970) . A .L . STEINER, J .A . FERRENDELLI and D .M . KIPNIS . J . Biôl . Chem . 247, 1121-1124 (1972) . A .L . STEINER, R .E . WEHMANN, C .W . PARKER and D .M . KIPNIS . In Advances in P, Greengard, G .A . Robison C clic Nucleotide Research, Vol . 2, p . 51-61 . an . ao ett , tors, Raven Press, N,Y . (1972) . G . KRISHNA, B . WEISS and B .B . BRODIE . J, of Pharmacology and Experimental Therapeutics , 163, 379-385 (1968) . O .H . LOWRY, M .J . ROSEBROUGH, A .L . FARR and R .J . RANDAL . J . Biol . Chem . 193, 265-270 (1951) . G .W . SNEDECOR and W .G . COCHRAN . Statistical Methods, 6th Edition . The 36 Iowa State University Press, Ames In Advances in C clic Nucleotide L .N . SIMON, D .A . SHUMAN and R,K . ROBINS . ors, Research , Vol . 3, p . 225-353 . P . Greengard an G . . Ro son, Raven Press, N .Y . (1973) . 91, 934-947 (1972) . S . REFETOFF, R . MATALON and M . BIGAZZI . Endocrinolo G .A . ROBISON, R .W . BUTCHER and E .W . SUTHERL yc is AMP , 1st Edition, p . 28, Academic Press, N .Y . and London (1971) . J .K . JONES, F . ISPiAIL-BEIGI and I .S . EDELMAN . J . Clin, Invest . 51, 24982501 (1972) . R .G . VAN INWEGEN, G . ALAN ROBISON, W .J . THOMPSON, K .J . ARMSTRONG and J .E . STOUFFER . J . Biol . Chem . 250, 2452-2456 (1975) .
