Cyclic GMP and cyclic GMP-dependent protein kinase in brown adipose tissue of developing rats

122

Biochimica et Biophysica Acta, 582 (1979) 122--131

© Elsevier/North-Holland Biomedical Press

BBA 28764 CYCLIC GMP AND CYCLIC GMP-DEPENDENT PROTEIN KINASE IN BROWN ADIPOSE TISSUE OF DEVELOPING RATS

JOSEF P. SKALA a,, and BRIAN L. KNIGHT b a Centre for Developmental Medicine, Department o f Paediatrics, University o f British Columbia, Vancouver, B.C., V5Z 1L7 (Canada) and b Medical Research Council Lipid Metabolism Unit, Hammersmith Hospital, London W12 OHS (U.K.)

(Received June 2nd, 1978) Key words: Cyclic GMP; Cyclic AMP; Catecholamine; Protein kinase; (Brown fat)

Summary In order to ascertain the possible involvement of cyclic GMP in the physiological regulation of the function and development of brown fat of the rat, we have determined its tissue concentration in vivo under a variety of conditions. The steady-state concentration of cyclic GMP in interscapular brown adipose tissue of late foetus was about 80 pmol per g fresh weight. The concentration gradually declined during the first 2 weeks after birth to reach 40 pmol/g fresh weight and then remained constant into adulthood. The cyclic GMP content of brown fat was decreased by chemical s y m p a t h e c t o m y and was increased after complete acclimatization of the animals to the cold. The activity of cyclic GMP-dependent protein kinase was also highest in tissue from newborn and cold-acclimatized rats. Both acute cold stress and injection of norepinephrine resulted in a significant but temporary increase in the concentration of cyclic GMP in brown fat, which was followed by a depression of the concentration below values in untreated animals. The concentration of cyclic AMP showed similar pattern of changes. Injection of phenylephrine was followed by a pronounced increase in the cyclic GMP c o n t e n t of brown fat, with little effect upon cyclic AMP. Injection of isoproterenol raised the content of cyclic AMP but not that of cyclic GMP. The ability of norepinephrine and phenylephrine to increase the concentration of cyclic GMP was abolished by pre-treatment of the animals with phenoxybenzamine, but not by pre-treatment with propranolol. Conversely, propranolol but n o t phenoxybenzamine abolished the effects of norepinephrine on the cyclic AMP content of the tissue. Thus we have established the responsiveness of the cyclic GMP content of * T o w h o m c o r r e s p o n d e n c e s h o u l d be s e n t . A b b r e v i a t i o n s : c y c l i c G M P , g u a m ) s i n e 3r, St-eyclic m o n o p h o s p h a t e ; monophosphate.

cyclic AMP, adenosine 3',5r-cyclic

123

brown fat to physiological and pharmacological stimuli and have evidence of the possible participation by cyclic GMP in the a-adrenergic stimulation and in the regulation of proliferative processes in the tissue.

Introduction

There is now much evidence to suggest that cyclic GMP is involved in the regulation of a variety of cell functions, such as neuronal excitation, membrane transport, enzyme secretion, proliferation and differentiation [1]. The cellular c o n t e n t of cyclic GMP can be altered under certain conditions by a wide range of hormones [2--5] and guanylate cyclase, which generates cyclic GMP, can be activated by various non-hormonal agents, including free fatty acids [6--9]. A protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) that can be stimulated by cyclic GMP has also been described (for review see ref. 1). However, the results obtained so far are not consistent enough to enable the true physiological role and mechanism of action of cyclic GMP to be defined. It is possible that cyclic GMP forms only a part of a more complex mechanism that involves other regulatory molecules, and does not act in the direct way that has been proposed for cyclic AMP. Thus, at this stage, experiments conducted under physiological conditions employing, where possible, specific natural stimuli, could be most useful in helping to establish the function of cyclic GMP. Brown adipose tissue is a well-characterized heat generating organ. Its thermogenic activity changes dramatically with ontogenic alterations in the animal's need for non-shivering thermogenesis [10]. Brown fat of rats rapidly proliferates and differentiates perinatally or during prolonged cold exposure of adult animals, and undergoes a gradual involution as the infants grow or the adults are returned to a thermoneutral environment [11]. The release and oxidation of free fatty acids, which are know to activate guanylate cyclase [9], is an essential part of the process of heat production in brown fat [12]. Both lipolysis and the developmental changes in brown fat are under hormonal control [13]. Thus the response of such a specialized tissue as brown fat to the natural stimuli that occur during maturation, growth and cold exposure could offer a good opportunity to examine the physiological significance of cyclic GMP in the regulation of cellular processes. In this paper we report our initial studies on the concentration of cyclic GMP and the activity of cyclic GMPdependent protein kinase in brown fat, and provide indirect evidence for the involvement of the nucleotide in the tissue's function and maturation. Both ~- and a-adrenergic agents have been reported to stimulate heat production in brown fat [14], whereas the adenylate cyclase system seems to be exclusively linked to /3-type receptors [15,16]. Since it has been suggested that catecholamine-induced accumulation of cyclic GMP is mainly associated with ~-adrenergic receptors [3], we have also studied the effect of a-adrenergic agonists and antagonists on the concentration of cyclic GMP in brown fat. Materials and Methods Animals. Albino Wistar rats (Woodlyn Laboratories, Ontario) were used throughout. Pregnancies were dated by vaginal smears and the infact animals

124 were delivered in our animal facility. Newborns were reduced to eight animals per litter and raised with their mother at 23°C in a 12 h light, 12 h dark cycle. Animals were weaned at day 30 and had free access to standard rat chow (Purina Chow, St. Louis, Mo.) and water. Adult rats (3 months old) were cold adapted by housing them for 6 weeks at 6 + 1~-'C before the experiment; intermixed littermates kept at 23°C served as controls. Short-term, acute cold exposure was precisely timed as specified in the figure legend. Chemical sympathect o m y by 6-hydroxydopamine was carried out as described previously [17]. Assay of cyclic nucleotides. After decapitation of the animals, interscapular pads of brown adipose tissue were rapidly excised, trimmed of extraneous tissue and immersed in liquid nitrogen. The time interval between the killing of the animal and the freezing of the tissue was carefully monitored and if exceeding 60 s the samples were discarded. Individual tissue samples of 25--100 mg fresh weight were stored at --80°C until used. Frozen pieces were weighed without thawing and homogenized in 2.0 ml of ice-cold 6% trichloroacetic acid using a Brinkman Instruments Polytron. Recovery tracer (cyclic[H3]GMP) was added to each sample, the homogenates spun at 3000 )~g for 15 rain at 4'~C and the supernatant neutralized with solid CaCO3 [18]. After repeated centrifugation the clear supernatants were evaporated in a shaking water bath at 75°C to decrease the volume to approx. 0.5 ml. The samples were then lyophilized in a Virtis apparatus equipped with a bulk vaccuum drum. The samples were dissolved in 2.0 ml 50 mM sodium acetate buffer, pH 6.2, and aliquots of 50--100 ~1 used for cyclic nucleotide determinations, and for recovery measurements. Cyclic GMP was determined by a radioimmunoassay according to Steiner et al. [19], using New England Nuclear (Boston, Mass.) assay kits. Cyclic AMP was determined by the protein binding assay of Gilman [20] using Amersham (Arlington Heights, Ill.) assay kits. Standard curves were prepared for each assay and the recoveries, which ranged between 65 and 85% recorded for each tissue sample. Samples were counted on a Beckman LS-9000 liquid scintillation counter and a Searle Instruments 1185 gamma counter, respectively. Since the cross-reactivity of the nucleotides with the antiserum and the binding protein is of crucial importance, we have tested it extensively. In our hands even a 104 times excess of the other nucleotide in the sample did not alter the value obtained for the competitive binding. Assay of cyclic GMP-dependent protein kinase. Brown fat was homogenized in 50 mM potassium phosphate buffer, pH 7.0, at 4°C using a Potter-Elvehjem homogenizer tube fitted with a motor-driven teflon pestle. The homogenate was centrifuged at 4°C for 30 min at 20 000 Xg and up to 50 ~g of supernatant protein assayed for the protein kinase activity. The assay mixture contained 50 mM potassium phosphate, pH 7.0, 10 mM MgC12, 0.3 mM ethyleneglycol-bis-([3-aminoethylether)-N,N'-tetraacetic acid, 0.5 mM isobutylxanthine, 0.2 mM [7-32p]ATP (30--50 cpm/pmol) and 6 mg/ml arginine-rich histone subgroup f3 (Sigma Cat. No. H4380) in a total volume of 50 ~1. The reaction was started by the addition of the enzyme and continued for 10 min at 30°C. 32p incorporated into protein was measured as described previously [21]. Materials. Cyclic nucleotides, 6-hydroxydopamine-HBr, norepinephrine (L-arterenol) and arginine-rich histone were purchased from Sigma Chem. Co.,

125

St. Louis, Mo.; phenylephrine-HC1 (Isuprel 0.5%) from Winthrop, L-propranolol (Inderal, 0.1%) from Ayerst Laboratories. Phenoxybenzamine-HC1 (Dibenzyline, 5%) was a kind gift from Smith Kline and French I.A.C. [7-32P]ATP was obtained from New England Nuclear {Boston, Mass.). Results Cyclic GMP in rat brown fat during development The steady-state concentration of cyclic GMP was determined in interscapular brown adipose tissue of rats of different ages. The tissue was always collected at the same time of day and the animals were disturbed as little as possible before killing. As shown in Fig. 1 the concentration of cyclic GMP ranged between 20 and 85 pmol/g fresh weight. Highest levels were observed in fetuses on day 21 of gestation, followed by a continuous decline during the first 2 postnatal weeks. The cyclic GMP content in brown fat of mature animals was 50--70% of that in newborns. Effects of cold acclimation and chemical sympathectomy Interscapular brown adipose tissue obtained from eigth fully cold-acclimatized adult rats contained 58 + 6 pmol cyclic GMP/g fresh weight. That was significantly (P < 0.05) more than 43 -+ 3 pmol/g fresh weight found in tissue from littermates kept in a thermoneutral environment. Functional stimulation of brown fat is mediated by the sympathetic nervous system [22]. Administration of 6-hydroxydopamine to infant rats results in a

80

,

,'/--v-c/

,

i

i

,%~-T- ¢ , '

i

l%

50

T

.-~

80

~

I I # ~ i i

E

)

f~ .

~

50

~

!~

~

~

2.o~ u~

O u

(3-

20

:E O

B' ~'

~'~ Age (days)

30'""~A

20

o-~.~__o

C

:E

~ l~

3'0"~' ~ 6 1'0 3~"~2~ -, Time ( rain )

Fig. 1. T h e " r e s t i n g " levels of c y c l i c G M P in i n t e r s c a p u l a r b r o w n a d i p o s e t i s s u e d u r i n g o n t o g e n i e d e v e l o p m e n t o f t h e r a t (B, b i r t h , v e r t i c a l b a r s r e p r e s e n t t h e S.E. of 1 2 - - 2 4 d e t e r m i n a t i o n s on 8 - - 1 6 a n i m a l s ) . Fig. 2. C y c l i c G M P ( e ) a n d c y c l i c A M P (o) c o n t e n t in b r o w n a d i p o s e t i s s u e o f 3 0 - d a y - o l d r a t s a f t e r e x p o sure of t h e a n i m a l s to 6 ° C ( A ) a n d a f t e r i n t r a p e r i t o n e a l i n j e c t i o n of 0.1 m g / 1 0 0 g b o d y w e i g h t nore p i n e p h r i n e (B). C, c o n t r o l s ; e a c h v a l u e r e p r e s e n t s t h e m e a n o f 4 - 6 d e t e r m i n a t i o n s on 2 - - 3 a n i m a l s . G r o u p e d v a l u e s d e t e r m i n e d at 5 a n d 10 m i n a n d t h o s e d e t e r m i n e d at 6 0 a n d 1 2 0 r a i n w e r e s i g n i f i c a n t l y d i f f e r e n t (P < 0 . 0 1 ) f r o m t h e c o n t r o l v a l u e s f o r b o t h n u c l e o t i d e s .

126 complete s y m p a t h e c t o m y of nerve terminals and preterminal fibres in brown fat [17]. Eight 1-week-old rats were injected with 6-hydroxydopamine-HBr (50 mg or free base/kg body weight) and their interscapular pads of brown fat assayed for cyclic GMP content 4 days later. Randomly interchanged littermates injected with saline served as controls. The tissue of the sympathectomized group contained 23 ± 5 pmol cyclic GMP/g fresh weight, whereas the tissue of control animalshad 47 + 6 pmol of cyclic GMP/g fresh weight (P < 0.01).

Effect o f cold exposure and of norepinephrine administration Acute exposure of rats to cold initiates a sequence of neurohormonal stimulatory responses which result in an increase in non-shivering heat production by brown adipocytes. 1-month-old rats reared at room temperature were exposed to 6 °C, killed at different times and the cyclic GMP content of brown adipose tissue assayed. As shown in Fig. 2A the concentration of cyclic GMP rose immediately after cold exposure. After 5 min the concentration gradually declined to reach values 60 min later which were well below the controls. When cyclic AMP was assayed in the same tissue extracts, a similar pattern emerged. In this case the initial increase lasted for 15--20 min and the depressed levels occurred after 30 rain exposure. Norepinephrine released from storage granules is assumed to mediate the stimulation of brown fat cells during cold exposure. We have therefore repeated the sequential assays of cyclic GMP content in brown fat, this time on animals at room temperature but injected with norepinephrine (0.1 mg/100 g body weight intraperitoneally). 1-month-old rats were again used and littermates injected with saline served as controls. As illustrated in Fig. 2B, a pattern of changes very similar to that occurring upon cold exposure emerged. Both cyclic GMP and cyclic AMP concentrations were increased 5--10 min after the norepinephrine administration and the levels of both nucleotides were depressed below the control values 30 min after the injections. Effect of ~- and [J-agonists and antagonists Norepinephrine is assumed to exert its effect on a target cell via both the a- and ~-adrenoreceptors. Phenylephrine, on the other hand, is believed to activate predominantly the a-receptors whereas isoproterenol stimulates mostly the ~-adrenoreceptors. Fig. 3 shows the influence of these specific adrenergic stimulants on the levels of cyclic GMP and cyclic AMP in brown adipose tissue of l-month-old rats. Isoproterenol-HC1 (0.1 mg/100 g body weight intraperitoneally) administration resulted in a small and transient increase in the concerttration of cyclic GMP and in a significant and longer-lasting elevation of the tissue's content of cyclic AMP (Fig. 3A). Conversely, the same dose of phenylephrine caused a pronounced increase in the concentration in brown fat of cyclic GMP but had little effect upon the concentration of cyclic AMP (Fig. 3B). Further experiments examined the in vivo effects of norepinephrine and phenylephrine after pre-treatment of the animals with the fl-adrenoreceptor blocker propranolol or the a-adrenoreceptor blocker phenoxybenzamine. Animals of the same age (1--3 months old) were randomly distributed into groups containing four animals each. Each group was injected with the same dose of

127

10 15

j

60

4C

i

A

B

?

o

I

'

l

30

-

.~

150

100

~

tm

50

L

~

°/o

<

100

cAMP

*

1.o

c

co

u

i

C

ll5

i

60

i

C

Time (rain)

ll5

h

60

u 50

Fig. 3. C y c l i c G M P ( e ) a n d c y c l i c A M P (~)) c o n t e n t in b r o w n a d i p o s e t i s s u e o f 3 0 - d a y - o l d rats a f t e r i n t r a peritoneal administration in i s o p r o t e r e n o l (A) and of phcnylephrine ( B ) in i d e n t i c a l d o s e s o f 0.1 m g / 1 0 0 g b o d y w e i g h t . (C, c o n t r o l s ; v e r t i c a l b a r s r e p r e s e n t t h e S . E . o f 8 - - 1 2 d e t e r m i n a t i o n s on 4--6 animals). Fig. 4. E f f e c t o f n o r e p i n e p h r i n e (NE) and phenylephrine ( P H E ) i n i d e n t i c a l d o s e s o f 0.1 m g / l O 0 g b o d y weight on the cyclic GMP (upper panel) and cyclic AMP (lower panel) content of interscapulax brown a d i p o s e t i s s u e o f rats p r e - t r e a t e d w i t h s a l i n e ( S A L ) , w i t h 0.4 r a g / 1 0 0 g b o d y w e i g h t o f p h e n o x y b e n z a m i n e ( P B A ) , or w i t h t h e s a m e d o s e o f p r o p r a n o l o l ( P R O ) . T h e f i g u r e s o n t o p i n d i c a t e t h e t i m e i n t e r v a l s in m i n u t e s . V a l u e s are e x p r e s s e d as p e r c e n t o f t h e c o n t r o l s i n j e c t e d w i t h s a l i n e o n l y . T h e m e a n o f t h e c o n trol group was assigned 100%. Vertical columns indicate the S.E., expressed in percent of the controls, of 4--12 determinations. An asterisk indicates P < 0.05 between the experimental group and the salinei n j e c t e d c o n t r o l s ; t i n d i c a t e s s i g n i f i c a n t (P < 0 . 0 5 ) e f f e c t o f p r o p r a n o l o l o r p h e n o x y b e n z a m i n e .

propranolol or phenoxybenzamine (0.4 m g / 1 0 0 g body weight intraperitoneally), or with an equivalent volume of saline. Subsequent administration (see Fig. 4) of identical doses of norepinephrine or phenylephrine were allowed to take effect for 5 or 10 rain, respectively, before the animals were killed and the cyclic nucleotide content of their interscapular brown adipose tissue assayed. Despite the rather large variations within each group, which would be expected from such in vivo studies, there were a number of significant differences between the groups (Fig. 4). Propranolol prevented the increase in cyclic AMP content of brown fat produced by norepinephrine and also abolished the slight increase observed with phenylephrine. It did not prevent the effects of norepinephrine or phenylephrine on the concentration of cyclic GMP in the tissue. Pre-treatment with phenoxybenzamine, on the other hand, had inconsistent effects upon the concentration of cyclic AMP but prevented the rise in cyclic GMP concentration produced either by norepinephrine or phenylephrine.

Cyclic GMP-dependent protein kinase activity in brown fat A cyclic GMP-dependent protein kinase has recently been purified from lung [ 2 3 - - 2 6 ] , heart [27] and aorta [28], and has been detected in extracts from a variety of tissues [29]. Cyclic GMP-dependent and cyclic AMP-dependent protein kinases can also be activated by cyclic AMP and cyclic GMP, respectively, but a much higher concentration is required to elicit the same effect as the more specific nucleotide [ 2 3 - - 2 6 , 2 9 , 3 0 ] . Thus after assaying a number of full-

128 TABLE I CYCLIC GMP-DEPENDENT PROTEIN AND COLD-ACCLIMATIZED RATS

KINASE

ACTIVITY

IN B R O W N

FAT OF NEWBORN,

ADULT

T h e 2 0 0 0 0 X ~ s u p e r n a t a n t f r a c t i o n f r o m i n t e r s c a p u l a r b r o w n a d i p o s e t i s s u e w a s p r e p a r e d as d e s c r i b e d i n Materials and Methods. Up to 50 pg of the extract was assayed for protein kinase activity in the presence o f a b s e n c e o f 0 . 3 # M c y c l i c A M P o r c y c l i c G M P (see M a t e r i a l s a n d M e t h o d s ) . R e s u l t s are g i v e n f o r t h e increase in kinase a c t i v i t y elicited b y each n u c l e o t i d e a n d f o r the relative ratio o f cyclic G M P a c t i v a t i o n to cyclic AMP activation. Unstimulated activities were within the range of 20--40 pmol/min per mg of p r o t e i n . V a l u e s are t h e m e a n ± S . E . o f 6 - - 9 d e t e r m i n a t i o n s c a r r i e d o u t o n t h r e e s e p a r a t e p o o l s o f t i s s u e s . Increase in protein kinase activity (pmol/rnin per mg protein) elicited by

Newborn rats Adult rats kept at 23°C A d u l t rats k e p t at 6°C for 6 weeks

Cyclic GMP

Cyclic AMP

ll

41 + 1 32 ± 7 53 ± 1 3

+ I 2 ± 1 * 13 ± 2

Cyclic GMP/cyclic AMP (%)

2 5 +- 2 5 -+ 1 * 26 ± 6

* S i g n i f i c a n t l y (P < 0 . 0 5 ) l o w e r t h a n n e w b o r n o r c o l d - a c c l i m a t i z e d .

range dose vs. response curves, we have employed a concentration of cyclic nucleotide (0.3 pM) which can activate its specific kinase but has negligible effect upon the other. A similar concentration was used by Kuo [31] to study the development of the two kinases in tissues of guinea pig. As a safeguard against cross-activation we have always compared the increase in kinase activity produced by 0.3 pM cyclic GMP with that produced by 0.3 pM cyclic AMP. The greatest ratio of cyclic GMP activation to that elicited by cyclic AMP was achieved with potassium phosphate as assays buffer, with 0.2 mM ATP as the phosphoryl group donor, and with arginine-rich histone as the protein substrate. Cyclic GMP activation in the crude tissue extracts did not require the presence of exogenous modulator protein or high concentration of magnesium, and was reduced if the extract was frozen and thawed. Table I shows that there was a greater increase in protein kinase activity on the addition of 0.3 pM cyclic GMP to extracts of brown fat from newborn and cold-adapted rats than with tissues from adult, warm-adapted rats. The ratio of cyclic GMP activation to cyclic AMP activation was also significantly increased, which indicates that the cyclic GMP was activating a true cyclic GMP-dependent protein kinase with a relatively greater activity in brown fat from newborn and fully cold-acclimatized rats. Discussion One of the ways to approach the elucidation of the biological importance of cyclic GMP is to try to associate changes in its tissue concentration with the actions of hormones and other agents that are known to modify specific aspects of cellular function. In this respect brown fat provides a very suitable model. The main, and probably the only, function of brown fat is to produce heat, and both the immediate response to cold and the ontogenic changes in its morphological and biochemical composition are under hormonal control [13].

129 Moreover, it is possible to effect pronounced changes in the physiological pattern of development by relatively simple changes in experimental conditions such as cold and warm exposure and s y m p a t h e c t o m y . These characteristics have proved extremely useful in studying the role of cyclic AMP in the mediation of hormonal effects [32--35]. The observation reported here on the response of cyclic GMP- and cyclic GMP-dependent protein kinase to physiological and pharmacological stimuli indicate that brown fat could be equally valuable model system in the studies of mechanisms of cellular regulations involving cyclic GMP. In order to establish the physiological significance of cyclic GMP in brown fat the presently reported experiments have examined the effects upon the tissue in vivo. Exposure of rats to cold produced an immediate rise in the tissue concentration of cyclic GMP. This suggests that cyclic GMP could play some part in the mechanism by which brown fat increases its rate of heat production upon cold exposure [36] and provides evidence of a physiological change in the concentration of cyclic GMP in response to a specific environmental stimulus. Administration of norepinephrine mimicked the effect of cold exposure upon the concentrations of cyclic AMP and cyclic GMP in brown fat. This again confirms that norepinephrine mediates the effects of cold upon the tissue. Injection of rats with the a-adrenergic agonist phenylephrine produced an increase in the cyclic GMP content of brown fat, whereas injection of the ~-adrenergic agonist isoproterenol produced an increase in the cyclic AMP content. Furthermore, the increases in the concentration of cyclic GMP, b u t not those of cyclic AMP, produced by norepinephrine and phenylephrine were prevented by the a-adrenoreceptor antagonist phenoxybenzamine. Despite the well-appreciated difficulty in interpretation of in vivo experiments, it seems reasonable to conclude from such observations that the cyclic GMP content of brown fat is influenced by a-adrenergic stimulation of the tissue. Accumulation of cyclic GMP in some other tissues has also been related to catecholamine stimulation of a-adrenergic receptors [3,37,38]. The present results with isoproterenol and the ~-blocker propranolol confirm the exclusively ~-adrenergic characteristics of the adenylate cyclase receptor in brown fat. Our observation on the link between cyclic GMP and the a-adrenergic receptor offers a possible explanation of the reports that heat production in brown fat can be increased by a-adrenergic agents [14] as well as by ~-adrenergic stimulation o f cyclic AMP production and lipolysis. In this respect it is interesting that cyclic GMPdependent protein kinase has been shown to activate hormone-sensitive lipase [39]. However, other studies failed to show any correlation between cyclic GMP content and lipolysis in white adipose tissue [40], so any conclusions a b o u t this apparent contradiction would be premature. The processes of proliferation and differentiation in brown adipocytes are very active perinatally. There then follows a short period of maximal activity and functional capacity which soon gives way to a period of de-differentiation and gradual involution. The latter can be prevented or reversed by prolonged cold exposure of the animals [11]. The high steady-state concentration of cyclic GMP in brown fat and the increased activity of cyclic GMP-dependent protein kinase observed during the early stages of development and upon cold acclimatization thus seem to correspond with periods of high proliferative

130

activity. Moreover, impairment of the sympathetic nervous system, which results in degenerative changes in brown fat of infact rats [17], causes a reduction of the concentration of cyclic GMP in the tissue. Involvement of cyclic GMP in developmental processes [41], and in the proliferation of hepatocytes [2] and spleen cells [42] had previously been proposed. The cause of the high concentration of cyclic GMP in brown fat around birth and during both acute and chronic cold exposure cannot be ascertained from the present experiments. Reports that the activity of guanylate cyclase is increased during rapid growth of the heart [43], and that the activity of the low K m phosphodiesterase for cyclic GMP is also increased in foetal lung [31] might indicate that the concentration of cyclic GMP in brown fat during early development is related to a stimulation of guanylate cyclase rather than to a low activity of the phosphodiesterase. There is much data to suggest that hormonal effects upon the activity of guanylate cyclase are brought about through an indirect mechanism. Free fatty acids released by activation of hormone-sensitive lipase [9], or the "feedback regulator" described in white fat by Ho and Sutherland [7] and known to activate guanylate cyclase [8] could possibly be involved in such an indirect stimulation of guanylate cyclase in brown fat. However, the observation that the concentration of cyclic GMP eventually falls below the control values after the initial rise produced by noreadrenaline or cold exposure indicates that the phosphodiesterase can also be activated and may be important for the regulation of the cyclic GMP content of brown fat. Acute cold exposure of rats initiates both an immediate increase in the rate of heat production in brown fat [36] and the trophic changes which become apparent during prolonged exposure [11]. The immediate increase in the concentration of cyclic GMP could replated to the stimulation of either or both of these responses. The coincidence of the changes in cyclic GMP content and cyclic GMP-dependent protein kinase activity with the changes in the rate of proliferation of the tissue certainly suggests a possible involvement of the nucleotide in the trophic stimulation of the tissue, and at the same time it confirms the usefulness of brown fat as a model tissue for studying the physiological actions of cyclic GMP. Acknowledgments We thank Mr. Salim Hassanali for his excellent technical assistance. This work was supported by Canadian Medical Research Council grant No. MA-5659 to J.P.S. who is a recipient of the Canadian Medical Research Council Scholarship. References 1 G o l d b e r g , N.D. a n d H a d d o x , M.K. ( 1 9 7 7 ) A n n u . Rev. B i o e h e m . 4 6 , 8 2 3 - - 8 9 6 2 M i u r a , Y., Iwai, H., S a k a t a , R., O h t s u k a , H., E l h a n a n , E., K u b o t a , K. a n d F u k u i , N. ( 1 9 7 6 ) J. Biochem. 80, 291--297 3 P o i n t e r , R . H . , B u t c h e r , F . R . a n d F a i n , J.N. ( 1 9 7 6 ) J. Biol. C h e m . 2 5 1 , 2 9 8 7 - - 2 9 9 2 4 Illiano, G., Tell, G . P . E . , Siegel, M.I. a n d C u a t r e c a s a s , P, ( 1 9 7 3 ) P r o c . N a t l . A c a d . Sci. U.S. 70, 2 4 4 3 2447

131

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

G o l d f a r b , R . D . a n d G l e n n , T.M. ( 1 9 7 6 ) E u r . J. P h a r m a c o l . 3 7 , 6 3 - - 7 0 M i t t a l , C . K . a n d M u r a d , F. ( 1 9 7 7 ) J. C y c l i c N u c l . Res. 3 , 3 8 1 - - 3 9 1 H o , R . J . a n d S u t h e r l a n d , E.W. ( 1 9 7 1 ) J. Biol. C h e m . 2 4 6 , 6 8 2 2 - - 6 8 2 7 A s a k a w a , T., R u ~ , J., S n y d e r , R . , S c h e i n b a u m , I., R u s e l l , R . T . a n d H o , R . J . ( 1 9 7 5 ) F e d . P r o c . 3 4 , 616 W a l l a c h , D. a n d P a s t a n , L ( 1 9 7 6 ) J. Biol. C h e m . 2 5 1 , 5 8 0 2 - - 5 8 0 9 H a h n , P. a n d S k a l a , J.P. ( 1 9 7 8 ) in R e v i e w s in P e r i n a t a l M a d i c i n e (ScarPelli, E.M. a n d C o s m i , E.V., eds.), Vol. 2, p p . 3 6 3 - - 3 8 3 , R a v e n Press, N e w Y o r k B a r n a r d , T. a n d S k a l a , J.P. ( 1 9 7 0 ) in B r o w n A d i p o s e Tissue ( L i n d b e r g , O., e d . ) , p p . 3 3 - - 7 2 , Elsevier, New York D r a h o t a , Z. ( 1 9 7 0 ) in B r o w n A d i p o s e Tissue ( L i n d b e r g , O., ed.), p p . 2 2 5 - - 2 4 4 , Elsevier, N e w Y o r k S k a l a , J.P. a n d H a h n , P. ( 1 9 7 4 ) Int. J. B i o c h e m . 5, 9 5 - - 1 0 6 H o r w i t z , B . A . a n d H o r o w i t z , J.M. ( 1 9 7 7 ) E x p e r i e n t i a 3 3 , 1 1 2 6 - - 1 1 2 8 S k a l a , J.P., H a h n , P. a n d B r a u n , T. ( 1 9 7 0 ) L i f e Sci. 9, 1 2 0 1 - - 1 2 1 2 P e t t e r s s o n , B. a n d Vallin, I. ( 1 9 7 6 ) E u r . J. B i o c h e m . 6 2 , 3 8 3 - - 3 9 0 S k a l a , J.P. a n d H a h n , P. ( 1 9 7 7 ) C a n . J. P h y s i o l . P h a r m a c o l . 5 5 , 2 7 2 - - 2 7 8 T i h o n , C., G o r e n , M.B., S p i t z , E. a n d R i c k e n b e r g , H . V . ( 1 9 7 7 ) A n a l . B i o c h e m . 8 0 , 6 5 2 - - 6 5 3 S t e i n e r , A . L . , P a r k e r , C.W. a n d K i p n i s , D.M. ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 1 1 0 6 - - 1 1 1 3 G i l m a n , A . G . ( 1 9 7 0 ) P r o c . N a t l . A c a d . Sci. U.S. 6 7 , 3 0 5 - - 3 1 2 S k a l a , J.P. a n d K n i g h t , B.L. ( 1 9 7 5 ) B i o c h i m . B i o p h y s . A c t a 3 8 5 , 1 1 4 - - 1 2 3 S c h o n b a u m , E., S t e i n e r , G. a n d Sellers, E . A . ( 1 9 7 0 ) in B r o w n A d i p o s e Tissue ( L i n d b e r g , O., ed.), p p . 1 7 9 - - 1 9 6 , Elsevier, N e w Y o r k K u o , J . F . , K u o , W.N., Shoji, M., Davis, C.W., S e a r y , V . L . a n d D o n n e l l y , T.E. ( 1 9 7 6 ) J. Biol. C h e m . 251, 1759--1766 Gill, G . N . , H o l d y , K . E . , W a l t o n , G.M. a n d K a n s t e i n , C.B. ( 1 9 7 6 ) P r o c . Nat]. A c a d . Sci. U.S. 73, 3918--3922 L i n c o l n , T.M., Dills, W.L. a n d C o r b i n , J . D . ( 1 9 7 7 ) J. Biol. C h e m . 2 5 2 , 4 2 6 9 - - 4 2 7 5 Shoji, M., P a t r i c k , J . G . , Davis, C.W. a n d K u o , J . F . ( 1 9 7 7 ) B i o c h e m . J. 1 6 1 , 2 1 3 - - 2 2 1 Shoji, M., P a t r i c k , J . G . , Tse, J. a n d K u o , J . F . ( 1 9 7 7 ) J. Biol. C h e m . 2 5 2 , 4 3 4 7 - - 4 3 5 3 K u o , J . F . ( 1 9 7 4 ) P r o c . N a t l . A c a d . Sci. U.S. 7 1 , 4 0 3 7 - - 4 0 4 1 K u o , J . F . , M i y a m o t o , E. a n d R e y e s , P.L. ( 1 9 7 4 ) B i o c h e m . P h a r m a c o l . 2 3 , 2 0 1 1 - - 2 0 2 1 R e i m a n n , E.M., Walsh, D . A . a n d K r e b s , E . G . ( 1 9 7 1 ) J. Biol. C h e m . 2 4 6 , 1 9 8 6 - - 1 9 9 5 K u o , J . F . ( 1 9 7 5 ) P r o c . N a t l . A c a d . Sci. U.S. 7 2 , 2 2 5 6 - - 2 2 5 9 S k a l a , J.P., D r u m m o n d , G.I. a n d H a h n , P. ( 1 9 7 4 ) B i o c h e m . J. 1 3 8 , 1 9 5 - - 1 9 9 S k a l a , J.P. a n d K n i g h t , B.L. ( 1 9 7 7 ) J. Biol. C h e m . 2 5 2 , 1 0 6 4 - - 1 0 7 0 K n i g h t , B.L. a n d S k a l a , J.P. ( 1 9 7 7 ) J. Biol. C h e m . 2 5 2 , 5 3 5 6 - - 5 3 6 2 S k a l a , J.P. a n d K n i g h t , B.L. ( 1 9 7 8 ) C a n . J. B i o c h e m . , in t h e p r e s s H i m m s - H a g e n , J. ( 1 9 7 6 ) A n n u . R e v . P h y s i o l . 3 8 , 3 1 5 - - 3 5 1 W o j c i k , J . D . , G r a n d , R . J . a n d K i m b e r g , D.V. ( 1 9 7 5 ) B i o c h i m . B i o p h y s . A c t a 4 1 1 , 2 5 0 - - 2 6 2 O ' D e a , R . F . a n d Z a t z , M. ( 1 9 7 6 ) P r o c . N a t l . A c a d . Sci. U.S. 7 3 , 3 3 9 8 - - 3 4 0 2 K h o o , J . C . , S p e r r y , P.J., Gill, G . N . a n d S t e i n b e r g , D. ( 1 9 7 7 ) P r o c . N a t l . A c a d . Sci. U.S. 7 4 , 4 8 4 3 - 4847 F a i n , J . N . a n d B u t c h e r , F . R . ( 1 9 7 6 ) J. C y c l i c N u c l . Res. 2, 7 1 - - 7 8 Davis, C.W. a n d K u o , J , F . ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A c t a 4 4 4 , 5 5 4 - - 5 6 2 D i a m a n t s t e i n , T. a n d U l m e r , A. ( 1 9 7 5 ) E x p . Cell Res. 9 3 , 3 0 9 - - 3 1 4 V e s e l y , D . L . , C h o w n , J. a n d L e v e y , G.S. ( 1 9 7 6 ) J. Mol. Cell. C a r d i o l . 8, 9 0 9 - 9 1 3