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The role of polyamines in β-adrenergic stimulation of calcium influx and membrane transport in rat heart
j Mol Cell Cardio120, 789-799 (1988)
The Role o f P o l y a m i n e s in ~-Adrenergic Stimulation o f Calcium Influx and M e m b r a n e T r a n s p o r t in Rat Heart Chien-Chung Fan and Harold Koenig*
Neurology Service, VA Lakeside Medical Center and the Department of Neurology, Northwestern University Medical School, Chicago, IL 60611, USA (Received 26 September 1986, acceptedin revisedform 6 May 1988) C.-C. FAN ANDH. KOI~NIG.The Role of Polyamines in//-Adrenergic Stimulation of Calcium Influx and Membrane Transport in Rat Heart. Journalof Molecularand CellularCardiology(1988) 20, 789--799. The/~-adrenergic agonist 1-isoproterenol induced an early (< 1 rain) stimulation of endocytosis, amino acid transport and hexose transport, monitored by the temperature-sensitive uptake of horseradish peroxidase, ~-aminoisobutyrate and 2-deoxyglucose, respectively, in rat ventricle cubes. This stimulation was time- and concentration-dependent and was maximum at 10-sM isoproterenol. The /~-adrenergic antagonist propranolol blocked isoproterenol stimulation of membrane transport, thereby confirming/Ladrenoceptor mediation; 2.5 mM EGTA, 1 mM LaC12 and 100 ,UMverapamil blocked the hormonal response without affecting basal transport. The calcium ionophore A23187 caused an acute stimulation of endocytosis, hexose and amino acid transport. Isoproterenol rapidly (< 30 s) stimulated 45Ca2 + influx. These data suggest that stimulus-response (stimulus-"transport") coupling is meditated by a rise in cytosolic Ca 2+ concentration. A rapid ( < 30 to 60 s) increase in ornithine decarboxylase (ODC) activity, followed by an early ( < 1 to 2 min), sustained increase in putrescine, spermidine and spermine concentrations was evoked by 10-7M isoproterenol. The ODC inhibitor ~-difluoromethylornithine (DFMO, 5 mM) suppressed the isoproterenol-induced increase in ODC and polyamine levels and the stimulation of 45Ca influx, endocytosis, hexose transport, and amino acid transport. Putrescine (0.5 m~) negated DFMO inhibition and restored the increase in polyamines, '~SCa influx, endocytosis, and transport of hexose and amino acid. These data suggest that polyamine synthesis is involved in isoproterenol stimulation of Ca 2+ influx and membrane transport functions in ventricular myocardium. These findings are consistent with a model for signal transduction and stimulu~response coupling in which polyamines function as intracellular messengers to generate cytosolic Ca 2+ signals by stimulating Ca 2 + influx. KEy WOROS: Isoproterenol; /~-Adrenergic agonist; /~-Adrenoceptor; Ornithine decarboxylase; Polyamines; Putrescine; Spermidine; Spermine; Ca2+influx; Membrane transport; Endocytosis; Amino acid transport; Hexose transport; Signal transduction.
Introduction /~-Adrenergic modulation of voltage-sensitive c a l c i u m c h a n n e l s in m y o c a r d i a l cells initiates a slow i n w a r d c u r r e n t c a r r i e d m a i n l y b y C a z + t h a t p r o f o u n d l y influences c a r d i a c f u n c tion a n d constitutes the m o s t i m p o r t a n t neuroregulatory control over heart action [29]. C a c h a n n e l m o d u l a t i o n a n d i n c r e a s e d C a influx a r e t h o u g h t to be m e d i a t e d b y a n e l e v a t i o n o f i n t r a c e l l u l a r c A M P [34] a n d p r o t e i n p h o s p h o r y l a t i o n [ 3 / ] , l e a d i n g to a n i n c r e a s e in the a v e r a g e n u m b e r o f f u n c t i o n a l C a c h a n n e l s p e r cell, a n d also c h a n g e s in the t i m e course o f b o t h a c t i v a t i o n a n d i n a c -
t i v a t i o n o f C a c h a n n e l s [-2, 30]. H o w e v e r , the m o l e c u l a r m e c h a n i s m s u n d e r l y i n g the c o n t r o l o f C a c h a n n e l a c t i v i t y in c a r d i a c muscle cells are still u n e l u c i d a t e d . R e c e n t studies in o u r laboratory have shown that a transient increase in o r n i t h i n e d e c a r b o x y l a s e ( O D C ) , the first a n d r a t e - l i m i t i n g e n z y m e o f the p o l y a m i n e s y n t h e t i c p a t h w a y , a n d a rise in polya m i n e levels a r e e a r l y c e l l u l a r events in m o u s e kidney cortex following receptor activation by t h e / / - a d r e n e r g i c a g o n i s t 1-isoproterenol [-16], a s well as b y testosterone [17], t r i i o d o t h y r o n i n e [-8], a n d insulin [9]. T h i s p o l y a m i n e synthesis has b e e n f o u n d to be essential for
* Please send reprint request to : Professor Harold Koenig, Neurology Service, VA Lakeside Medical Center, 333 E. Huron Street, Chicago, IL 60611, USA. 0022-2828/88/090789 + 11 $03.00/0
9 1988 Academic Press Limited
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C.-C. Fan and H. K o e n i g
hormonal stimulation of Ca z+ fluxes and several Ca 2 +-dependent membrane transport processes (reviewed in [20]). We report here experiments suggesting that isoproterenol stimulation of Ca z + fluxes and Ca 2 +dependent membrane transport processes in rat heart appears to be mediated by rapid polyamine synthesis. A brief account of some of these results has appeared in abstract form
[53. Methods
Incubations Female Sprague-Dawley rats ( ~ 150 to 200 g) were used for these studies. Animals were killed by decapitation and hearts were rapidly excised and placed in an ice-cold physiological salt solution (PSS) containing (in raM) : Na 122, KC1 5, CaC12 2.7, MgSO4 1.2, N a z H P O , ~ 0.6, glucose 6, H E P E S buffer (pH 7.4) 39. Ventricles were dissected free and cut into .-~0.5 m m cubes with a McIlwain tissue chopper. Incubations were carried out in 25-ml plastic beakers in PSS with appropriate additions at 37~ under 95% O z in a Dubnoff shaking incubator.
Rates of endocytosis, hexose and amino acid transport Endocytosis, amino acid transport and hexose transport were measured by monitoring the temperature-sensitive uptake of horseradish peroxidase (HRP), e-[ 1-x4C]aminoiso butyrate (AIB), and 2-deoxy-D-[1-aH]-glu cose (DG) in heart cubes as previously described for kidney slices [7, 15]. H R P (1 mg/ml), 14C-AIB (0.1mM, 4/~Ci/ml) and 3H-DG (0.1 raM, 1 #Ci/ml) were added to the incubation medium. Incubations were terminated by placing beakers on ice and washing ventricle cubes three times with fresh medium by centrifugation. Corrections for nonspecific uptake were made by subtracting the amount of tracers taken up during parallel incubations at 0~ Slices were sonicated in H 2 0 for measurement of protein, H R P and radioactivity (by scintillation spectrometry).
Rate of 45Ca influx 45Ca influx was measured by a modification of the method of Lodge et al. [22]. Heart
cubes were pre-incubated briefly (5 min) in PSS with appropriate additions, transferred to fresh media containing 45Ca (1 to 2 g C i / ml) and isoproterenol or vehicle, and incubated with constant shaking under 95% 0 2 - 5 % CO2 at 37~ for 30, 60 and 90s. Incubations were terminated by chilling on ice, and aliquots of incubates were washed twice in 3 vols of ice-cold PSS by centrifugation. The pelleted heart cubes were resuspended in PSS containing 7.5mM Ca z+ and 6 m~ EGTA, and shaken for 1 h at 0~ in order to remove most of the extracellular 45Ca2+. To correct for residual extracellular 45Ca, heart cubes were pre-incubated and incubated at 0~ as described above, and subjected to the same wash procedure. The 0~ uptakes were then subtracted from the 37~ uptakes. In principle, the exchange and removal of extracellular free and bound 45Ca2+ should be facilitated by the large Ca 2 + buffering pool provided in this wash solution, while cooling to 0~ will decrease transmembrane Ca 2 + movement without greatly impeding the diffusion of extracellular Ca, and thus preserve much of the intracellular 45Ca [1, 22, 35]. Cells injured during sectioning are believed to become isolated owing to a reduction in intercellular junction permeability induced by the membrane lesion [4], and the C a - E G T A wash procedure should deplete such damaged cells of 45Ca. In the present experiments 0~ uptake was timedependent and insensitive to additions, and was generally 25 to 45% of the 37~ uptake in basal or DEMO-inhibited heart, and 15 to 22% of that in isoproterenol-stimulated heart. The initial rates of 45Ca influx in unstimulated heart cubes were comparable to these obtained by Lodge et al. [22] in thin canine ventricle sheets, and in the isolated, perfused rat heart [10, 13].
Analytical methods Protein was determined according to Lowry et al. [23], using bovine serum albumen as standard. H R P was assayed according to Steinman and Cohn [32]. Polyamines were extracted by homogenizing tissue samples in cold 0.2 ~ perchloric acid, and removing the insoluble residue by centrifugation. Polyamines were measured by spectrophotofluori-
Role of Polyamlnes in p-Adrenergic Effects
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FIGURE 1. Effect ofisoproterenol on the time course of (a) HRP, (b) DG and (c) A1B uptake by rat heart in vitro. Ventricular cubes were pre-incubated in PSS for 10 rain at 37~ At zero time the medium was replaced by fresh medium coataining HRP (1 mg/ml), [ 14 C]AIB (0.1 mM, 4#Ci/ml), [ 3 H]DG (0.1 raM, 1/~Ci/ml), and 10- ? Misoproterenol (O . . . . C)) or no hormone ( 0 - 0 ) . Tissue samples were removed for study at each time point of incubation commencing at 0.5 rain. Data were corrected for nonspecific uptake as described in Methods. Results are means _ S.E.M.(n = 3). * P < 0.05 (Student's t-test). m e t r y o f the d a n s y l a t e d derivatives after separation by thin-layer chromatography [27]. Recoveries of polyamines, as assessed with a n i n t e r n a l s t a n d a r d , a v e r a g e d 70%, a n d d a t a were corrected for these recoveries. O r n i t h i n e d e c a r b o x y l a s e activity was determ i n e d b y a modification [17] of the m e t h o d o f D j u r h u u s [3]. Tissue samples were h o m o g e nized in a p p r o x i m a t e l y 5 vols of 50mM s o d i u m - p h o s p a t e buffer, p H 7.2, c o n t a i n i n g 5 mM dithiothreitol, 0.1 m ~ e t h y l e n e d i a m i n e
tetraacetic acid ( E D T A ) a n d 40 mM p y r i d o x a l phosphate. H o m o g e n a t e s were centrifuged at 12000 • g for 20 min at 4~ a n d the pellets were discarded. S u p e r n a t a n t (0.1ml) was a d d e d to 0.1 ml o f i n c u b a t i o n m e d i u m consisting of 50mM s o d i u m - p h o s p h a t e buffer, p H 7.2, 5ram dithiothreitol, 200#M-pyridoxal phosphate, 0.1 mM L-ornithine, a n d 0.3/~Ci of 3 - 3 H ( n ) - o r n i t h i n e (20 C i / m m o l e -1, New E n g l a n d Nuclear). T h e tubes were i n c u b a t e d for 2 h at 37~ and then were cooled to 4~
792
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Incubation mixture (40/A) was applied to Whatman P-81 cation exchange paper, and paper strips were subjected to descending chromatography for 3 h with 0.1 M N H 4 O H to elute unreacted ornithine. The strips were dried and the spots corresponding to a putrescine standard detected by ninhydrin were cut out, placed in scintillation vials with 1 ml of deionized water, and heated at 50~ for 2 h. Aquassure scintillation fluid (10ml) (New England Nuclear) was added to each vial and the radioactivity was counted in a Beckman LS-250 liquid scintillation counter. Enzyme controls containing the complete assay mixture and boiled enzyme were run with each determination and the values were subtracted from the experimental values. O D e activity was expressed as pmol of putrescine H - 1. O D e activity was linear with time and protein in the range in which determinations were made. All results are presented as means _ standard errors. The statistical significance of differences between means was evaluated by Student's t-test.
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HRP, AIB and DG uptake The temperature-dependent uptake of H R P was linear between 0 and 10 min, and between 10 and 60 min, with the rate of uptake being two-fold greater during the first 10 min than during the subsequent 10 to 60
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3H-DG was purchased from Amersham (Arlington Heights, IL). L-[3-3H(n)]orni thine, 14C-AIB, 4Sea, and Aquasol came from New England Nuclear (Boston, MA). Horseradish peroxidase (Type II) and 1isoproternol came from Sigma Chemical Co. (St. Louis, MO). 0~-Difluoro-methylornithine was supplied by Merrell Dow Research Center (Cincinnati, OH), A23187 was obtained from Calbiochem-Behring (LaJolla, CA) and verapamil Dx-isopropyl-D-(Nmethyl-N-homoveratryl)-c<-amino-propyl-3, 4-dimethoxyphenylacetonitrile hydrochloride] from Knoll Pharmaceutical Co. (Whippany, NJ). Female albino SpragueDawley rats were from Holtzman (Madison, WI).
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F I G U R E 2. Effect of isoproterenol concentration on (a) HRP, (b) AIB and (c) DG uptake by rat heart. After a 10 min pre-incubation in PSS at 37~ ventricular cubes were placed in fresh medium containing HRP, [14C]AIB and [SH]DG and various concentrations of isoproterenol and incubated for 5 min at 37~ Hormonefree control uptake values were: HRP, 4 5 _ 3.7 ng/5 m i n / m g protein; DG, 4 1 3 + 3 1 . 5 pmol/5 min/mg protein; AIB, 151 + 19.4 pmol/5 min/mg protein. Additional details are given in Methods. Results are means + s.~.M. (n = 3). *, **, ***: P < 0.05, 0.01 and 0.0(31 (vs. control).
min (Fig. 1). This uptake varied linearly with solute concentration (1 to 10 mg/ml) consistent with fluid volume endocytosis. Isoproterenol increased the rate of H R P uptake by 50 to 120% at 5 min. This increase developed immediately and was readily detected at 1 min when a greater concentration of H R P (25 mg/ml) was used. Stimulation of H R P uptake varied with isoproterenol concentration and was greatest at 10 -8 M (Fig. 2). The temperature-sensitive uptake of AIB and DG by heart cubes was linear for about 2 min and then gradually declined over the duration of the incubation (Fig. 1). Isoproterenol increased t h e rate of AIB and DG
Role of Polyamines in ~-Adrenergic Effects 8O
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FIGURE 4. Effects ofisoproterenol on 4SCa influx in rat heart. Ventricular cubes were incubated at 37~ in PSS containing 4Sea without (O O) and with (Q) - - - Q) ) 10- v Misoproterenol. Results are means _+ S.E.M.(n = 3).
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FIGURE 3. Effect of E G T A , lanthanum, vcrapamil and A23187 on basal and isoprotcrcnol-stimulatcd
uptake of (a) HRP, (b) AIB and (c) DG by rat heart. Ventricular cubes were pre-ineubated at 37~ for 10 min in 2.5mM EGTA in Ca2+-free PSS, 1 mM lanthanum chloride (La3+), 100mM verapamil (VER), or [0•M A23187 in PSS containing 2.7mM Ca 2+, and then were incubated for 5 min in the same medium containing HRP, [14C]AIB, [3H]DG, 10-TM isoproterenol (m) or no hormone ([-]). Data are means _ S.E.M.(n = 3).
u p t a k e b y 40 to 100% at 5 min. Isoproterenol s t i m u l a t i o n o f A I B a n d D G u p t a k e was c o n c e n t r a t i o n - d e p e n d e n t a n d m a x i m u m at 10-SM (Fig. 2). The /Ladrenergic antagonist propranolol (10-TM) blocked the increase in H R P , A I B
a n d D G u p t a k e i n d u c e d b y 10-SM isoproterenol, b u t h a d no effect on basal u p t a k e of these substrates ( d a t a not shown). This confirms t h a t isoproterenol s t i m u l a t i o n of these m e m b r a n e t r a n s p o r t processes is m e d i a t e d b y ~ - a d r e n o r e c e p t o r activation. Figure 3 shows the effect o f E G T A , L a 3+, v e r a p a m i l a n d A23187 on basal a n d isoproterenol-stimulated H R P , A I B a n d D G uptake. T h e C a 2+ c h e l a t o r E G T A (2.5mM, no a d d e d C a 2+) and the C a antagonist L a 3 + (1 mM) caused a statistically insignificant decrease in b a s a l H R P , A I B a n d D G u p t a k e and abolished isoproterenol stimulated u p t a k e of these substrates into ventricle cubes. T h e Ca c h a n n e l blocker v e r a p a m i l (100/~M) in the presence of 2.7 mM C a 2+ did not alter basal u p t a k e o f H R P , A I B a n d D G , b u t abolished the isoproterenol response. I n contrast, the Ca i o n o p h o r e A23187 (10#M) i n d u c e d a two-fold increase in the u p t a k e of H R P , A I B a n d D G , a n d a d d i t i o n o f isoproterenol (10-SM) caused no further increase in this uptake. These experiments indicate that isoproterenol stimulation of endocytosis, hexose a n d a m i n o acid t r a n s p o r t requires extracellular Ca 2 +, C a 2 + b i n d i n g to extracellular sites, a n d Ca 2+ t r a n s p o r t in heart, as it does in kidney cortex [7].
"Sea Influx Isoproterenol (10-TM) i n d u c e d substantial increases (81 to 162%) in 45Ca into ventricle
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bation in the continued presence of the agonist [Fig. 5(a)]. All three polyamines rapidly increased to m a x i m u m levels at 5 min, and remained elevated during a 60 min incubation (Fig. 5). The rank order of increase of these constituents (percentage of control value) after 1 min of incubation with isoproterenol was: O D C (246%, P < 0.001) > putrescine (184%, P < 0.01) > spermidine (182%, P < 0.05) > spermine (128%, P > 0.05 < 0.1). Propranolol (10-6M) blocked the isoproterenol (10 -v~)-induced increase in O D C and polyamine levels in ventricle slices (data not shown). These data show that an increase in O D C activity and enhanced polyamine synthesis are very early /7-adrenoceptor-mediated events in isoproterenol-stimulated rat heart.
Role of polyamines in membrane transport
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e-Difluoromethylornithine (DFMO), a specific, irreversible, enzyme-activated inhibitor of O D C [26, 33], was used to assess the role of I00 polyamines in isoproterenol stimulation of il I [ I ii 1 m e m b r a n e transport functions. Figure 6 01 2 5 Io 60 shows that 5mM D F M O abrogated the Time of ineub0tion (rain) increase in endocytosis, hexose transport and FIGURE 5. lsoproterenol induces an acute increase amino acid transport induced by isoproin ODC activity and polyamine lavels in rat heart. Ventricular cubeswere incubated at 37~ with 10-v~ isopro- terenol. Putrescine (0.5m~), the product of terenol for various times and assayed for ODC and O D C activity, reversed the effect of D F M O polyamines as described in Methods. Zero-time control and restored the increase in endocytosis, values were: (a) ODC (O), 971-t-163 pmol/h/mg hexose transport and amino acid transport. protein; putrescine (C)), 0.143 -t- 0.026 nmol/mg protein; (b) spermidine, 0.66-I-0.14 mg protein; (c) D F M O blocked the isoproterenol-induced spermine, 1.93 -I-0.16 nmol/mg protein. Data are means increase in polyamine levels, and putrescine 4- S.E.M.(n = 3--4). repleted heart polyamines (Table 1). D F M O also inhibited basal membrane transport (not cubes during 30 to 90s incubations (Figs 4 shown) and decreased polyamine concentrations during a 15 min incubation (Table 1). and 7). Putrescine mimicked the effect of isoproterenol in that it stimulated membrane transODC activity andpolyamine port (not shown) and increased the levels of concentrations polyamines in rat heart during a 15 min incuIsoproterenol, 50 nmol/kg body wt intraperibation (Table 1). These results provide pertoneally, evoked a rapid ( < 2 rain) increase in suasive evidence for the view that rapid the concentrations of heart polyamines in vivo polyamine synthesis is involved in the acute (data not shown). Isoproterenol also induced effect of isoproterenol on membrane transport an early stimulation of O D C activity and an in rat heart. accumulation of polyamines in ventricle cubes in vitro (Fig. 5). O D C activity increased Role of polyamines in Ca z + influx rapidly after 10-TM isoproterenol to a maximum at 1 to 2 min and then declined but D F M O was also used to evaluate the role of still remained elevated after 60 min of incu- polyamines in isoproterenol stimulation of 200
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Role of Polyamines in [i-Adrenergic Effects
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FIGURE 6. DFMO blocks isoproterenol stimulation of endocytosis, amino acid transport and hexose transport in rat heart, and putrescine negates the DFMO effect. Ventricular cubes were pre-incubated for 10 min at 37~ in PSS with 5 mM DFMO, DFMO and 0.5 mM putrescine (PUT), or no additions. Cubes were transferred to fresh medium of the same composition containing HRP, [14C]AIB, [3H]DG, and 10- 7 ~ isoproterenol (ISO), or no hormone (control, C) and incubated for 5 min. Data are means ___S.E.M. (n = 3). Control uptake values (means +_ S.E.M.) were: (a) HRP, 29.3 _+ 2.8 ng/5 min/mg protein; (b) AIB, 142 -I- 18 pmol/5 min/mg protein; (c) DG, 216 -t- 23 pmol/5 rain/rag protein. *,**, P < 0.05, 0.01, (vs. control). ~', ~;~,P < 0.05, 0.01, (vs. isoproterenol).
Ca z + influx. DFMO blocked the stimulation of 4SCa influx evoked by isoproterenol during i n c u b a t i o n s o f 30 to 90 s (Fig. 7). P u t r e s c i n e n e g a t e d D F M O i n h i b i t i o n a n d p a r t i a l l y res t o r e d t h e i n c r e a s e i n 4 5 C a influx. D F M O d i d n o t a f f e c t 4 5 C a i n f l u x d u r i n g a 6.5 m i n i n c u bation; however, putrescine enhanced 45Ca i n f l u x d u r i n g a 6.5 m i n i n c u b a t i o n . T h e s e data support the inference that rapid polya m i n e s y n t h e s i s is i n v o l v e d i n i s o p r o t e r e n o l -
mediated heart.
s t i m u l a t i o n o f C a 2+ i n f l u x i n r a t
Discussion T h e d a t a p r e s e n t e d i n t h e first p a r t o f this paper have demonstrated that nanomolar concentrations ofisoproterenol induce a rapid (1-2 min) stimulation of endocytosis, amino
T A B L E 1. Effect of D F M O a n d putrescine on the rapid increase in heart polyamines induced by isoproterenol
Treatment No addition 10-7M isoproterenol + 5mM D F M O + 5 mM D F M O + 0.5 mM putrescine 5 mM D F M O 0.5 mM putrescine
Putrescine ( n m o l / m g protein)
Spermidine ( n m o l / m g protein)
Spermine ( n m o l / m g protein)
0.123 -I- 0.001 0.196 -I- 0.013 r 0.110 -t- 0.010 d
0.677 _-/-0.036 1.144 + 0.060 c 0.460 -I- 0.064 d
1.256 -I- 0.053 2.037 -I- 0.156 ~ 0.829 ___0.103 d
0.154 -t- 0.016 e 0.096 -I- 0.012" 0.120 _ 0.013
0.900 -t- 0.026 ~ 0.509 -I- 0.066 a 0.849 _ 0.057 a
1.627 -t- 0.023 f 0.883 -t- 0.056 b 1.596 _ 0.106 a
Heart slices were pre-incubated at 37~ for 10 min in PSS with 5 mM DFMO, 0.5 mM putrescine, DFMO and 0.5ram putrescine, or no addition, and then were incubated for 5 rain with or without 10-TM isoproterenol. Corrections for nonspecific uptake of putrescine were made by subtracting the 0~ uptake. Results are means __S.E.M. (n = 3). a,b,r p < 0.05, 0.01, 0.001 (vs. no addition), d: p < 0.001 (vs. isoproterenol). ~,f,g: P < 0.05, 0.01, 0.001 (vs. isoproterenol and DFMO).
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FIGURE 7. DFMO abolishes the isoproterenol-induced increment in '*SCa influx in rat heart, and putrescine negates DFMO inhibition. Ventricular cubes were preincubated for 5 min at 37~ with 10ram DFMO, DFMO and 1mM putrescine, 1 mM putrescine, or no agent, transferred to fresh medium of the same composition containing 4SCa and 10-vM isoproterenol or no hormone (control), and incubated for the given time periods. Data are means + s.E.~t. (n=3). **: P<0.01 (vs. control) ",~, "~r162 P<0.05, 0.01 (vs. isoproterenol); +, + + : P<0.05, 0.01 (vs. isoproterenol + DFmo). acid transport and hexose transport in rat ventricular myocardium. This m e m b r a n e transport response is mediated by fladrenoreceptor occupancy as it is elicited by the specific fl-adrenergic agonist isoproterenol, demonstrates h o r m o n e concentration dependence, and is abrogated by the fladrenergic antagonist propranolol. These experiments further show that isoproterenol stimulation of m e m b r a n e transport requires the presence of Ca 2+ in the extracellular medium, and appears to involve an increased inflow of 4 5 Ca 2 + . Enhanced Ca 2 + influx by isoproterenol has been previously reported during depolarization [28] and in the perfused beating heart [10, 13]. O u r results for basal 4~Ca uptake in rat heart cubes are in good agreement with those obtained in isolated perfused rat heart [10, 13~, and in thin canine heart sheets [22], when their results are expressed as Ca z+ uptake per min per mg protein. I n the work of H u n t e r et al. [13] and Gotzsche [10], the relative increase after isoproterenol stimulation was 46% and 39 to 53% at 1.5 x 10 -6 and 10-4M, as compared with 8 1 - 1 6 2 % at 10-7M isoproterenol in our
experiments. E n h a n c e d Ca 2+ influx appears to be important for the isoproterenol-induced m e m b r a n e transport response as it was abolished by the Ca 2 + chelator E G T A , the Ca 2 + antagonist La 3 +, and the organic Ca 2 + transport blocker verapamil. These data support the view that fl-adrenergic stimulation of cardiac myocytes increases cytosolic Ca 2+ concentration by enhancing the inflow of extracellular Ca 2+ across the sarcolemma. It is evident from these results that the rates of endocytosis, hexose transport and amino acid transport in ventricular m y o c a r d i u m are modulated by fl-adrenergic stimulation in a coordinate manner. These experiments have established that isoproterenol causes a rapid, fl-adrenoceptormediated stimulation of O D C activity and polyamine synthesis in rat ventricular myocardium. T h e early increase in heart O D C and polyamine levels is similar to that evoked by isoproterenol in mouse kidney cortex [16]. Previous studies have indicated that fladrenergic agonists increase heart O D C activity after a lag period of 1 to 4 h [6, 21, 36]. Synthesis of prostaglandins and cyclic A M P
Role of Polyamlnes in ~-Adrenerglc Effects
appears to be involved in the rapid stimulation of ODC activity and membrane transport functions by isoproterenol, as these responses are blocked by dexamethasone, aspirin, and 2',5'-dideoxyadenosine, inhibitors of arachidonate release, cyclooxygenase, and adenylate cyclase respectively, in mouse kidney cortex ([16, 20]; H. Koenig, A. D. Goldstone, C. Y. Lu, unpublished observations). In addition the prostaglandins PGA2 and PGE2, and dibutyryl-cyclic AMP have been shown to mimic the effects of isoproterenol in stimulating ODC activity and membrane transport processes [24]; H. Koenig, A. D. Goldstone, C.Y. Lu, unpublished observations). We have therefore suggested that this rapid stimulation of ODC activity likely represents a fl-receptor-mediated, Ca z +-, prostaglandin-, and cyclic AMPdependent activation of a latent ODC via a posttranslational process, possibly a rapidly reversible phosphorylation-dephosphorylation sequence involving O D C or an ODC regulating protein [16, 17, 20]. In addition, cytoskeletal participation appears to be important for isoproterenol stimulation of ODC activity and membrane transport functions as this stimulation is blocked by the microfilament-disrupting agent cytochalasin B and the microtubule-disrupting agent colchicine [20]; H. Koenig, A. D. Goldstone & C. Y. Lu, unpublished data). The timedependent decline in heart ODC activity in the continued presence of isoproterenol could reflect a desensitization effect resulting from uncoupling of the fl-receptor-adenylate cyclase complex [25"]. Guarnieri et al. [11, 12] have described an early fl-adrenoceptor-mediated increase in ODC in isolated, perfused rat heart which manifests Ca 2 +- and calmodulindependence and is independent of protein synthesis. In the present study the rank order of increase of polyamines at 1 rain was putrescine > spermidine > spermine. This rank order is consistent with the precursorproduct relationship observed in the ODCmediated pathway for polyamine synthesis [33]. Direct support for this inference comes from the finding that DFMO, a specific, irreversible, enzyme-activated, suicide inhibitor of ODC [26, 33], blocked the rapid isoproterenol-induced increase in polyamine
797
levels. Although the kinetic properties of ODC were not characterized, the observed ODC activity in isoproterenol-stimulated heart, ~-,30 pmol/min/mg protein, would appear to be too low to account for the observed increment in total polyamines, ,-, 145 pmol/min/mg protein. A similar disparity between ODC product and ODC activity is apparent in other rapid responses, including testosterone-J17] and isoproterenolstimulated kidney cortex [16"j, cryoinjured brain [18], and potassium depolarized brain synaptosomes [14]. Although the basis for this disparity is uncertain, its consistent occurrence in a variety of rapid cell responses to external stimuli strongly suggests that the catalytic activity of ODC in intact stimulated cells may be much greater than that measured in vitro in broken cell preparations or cytosolic extracts. These experiments provide direct evidence that rapid polyamine synthesis is involved in the isoproterenol-mediated increase in Ca 2+ influx and Ca 2 +-dependent membrane transport processes in ventricular myocardium, as it is in kidney cortex [16]. Thus D F M O suppressed isoproterenol stimulated polyamine synthesis and concurrently blocked the increase in 45Ca2 + influx, endocytosis, amino acid transport and hexose transport. Repletion of heart polyamines with putrescine negated D F M O inhibition and restored the increment in 4SCa2+ influx and Ca 2+d e p e n d e n t membrane transport functions. These data support the hypothesis that polyamines function as intracellular messengers to augment Ca 2 + in-flow across the sarcolemma. The consequent elevation in sarcoplasmic C a 2 + concentration would trigger the coordinate stimulation of endocytosis, hexose transport and amino acid transport and stimulate other Ca 2 +- (or Ca 2 +-calmodulin-) and polyamine-dependent reactions, e.g. excitation-cotraction coupling, in cardiac myocytes. Thus a recent study has implicated polyamines in the fl-adrenergic stimulation of heart contractility and rate [19]. The data described in this paper support the hypothesis that rapid ODC activation and polyamine synthesis' play a role in signal transduction and transmission and stimulusresponse coupling in heart cells. In this model transduction of the fl-adrenergic signal into
798
C.-C. Fan and H. K o e n i g
an intracellular message for the generation of a Ca 2 + signal involves a receptor- and Ca 2 +dependent activation of a latent O D C located in or near the sarcolemma. Transmission of the/~-adrenergic message is effected by polyamines initially synthesized in the subsarcolemmal microdomain. Newly synthesized polyamines may serve as messengers to generate Ca/+ signals by regulating Ca channel activity, Na/Ca exchange, and Ca 2 + influx by one or more of the following effects: (1) by displacing bound calcium via a cation exchange reaction; (2) by binding to anionic sites and acting as counterions to decrease surface charge and potential; (3) by modulating the phosphorylation states of proteins associated with the calcium channel or
calcium exchanger through polyaminedependent kinases or phosphatases. In this model the signaling action of polyamines is terminated when they bind to anionic sites or are degraded. It remains for future studies to elucidate the precise molecular mechanisms by which polyamines regulate transmembrane Ca 2 + movements.
Acknowledgements We thank Dr A. D. Goldstone and Mr C. Y. Lu for valuable assistance in several experiments, and Mrs T. Howell for the secretarial work. The research was supported by the Veterans Administration Research Service and N I H grants NS 18047 and H L 26835.
References 1 AARONSON,P., VANBREEMEN,R., LOUTZENHISER,R., KOLBER, M. A. A new mcthod for measuring the kinetics of transmembrane calcium-45 efflux in the smooth muscle of the guinea pig taenia coli. Life Sci 25, 1781 1790 (1979). 2 BEAN,B. P., Nowvmv, M. C., TSXEN,R. W. fl-Adrenergic modulation of calcium channels in frog ventricular heart cells. Nature 307, 371-375 (1984). 3 DJURHUUS,R. Ornithine decarboxylase assay based upon the retention of putrescine by a strong cation-exchange paper. Anal Biochem 113, 352-355 (1981). 4 DE MELLO,W. C. Modulation ofjunctional permeability. Fed Proc 43, 2692 2696 (1984). 5 FAN,C.-C., KOEmO, H. fl-Adrenergic stimulation of calcium fluxes and membrane transport processes in rat heart is mediated by polyamines. [Abstract] Fed Proc 43, 735 (1984). 6 FULLER,R. W., HEMRICK,S. K. Studies of noradrenergic influence on cardiac ornithine decarboxylase in rats. J Mol Cell Cardiol 10, 1031 1036 (1978). 7 GOLDSTONE,A. D., KOENIO,H., Lu, C. Y., TROUT,J. J. fi-Adrenergic stimulation evokes a rapid Ca 2+-dependent stimulation of endocytosis, hexose and amino acid transport associated with increased Ca 2+ fluxes in mouse kidney cortex. Biochem Biophys Res Commun 114, 913 921 (1983). 8 GOLDSTONE,A., KoEm% H., Lu, C. Y. Triiodothyronine (Ts) stimulation of Ca 2+ influx and membrane transport processes in mouse kidney cortex is mediated by polyamine synthesis. [-Abstract] Fed Proc 43, 735 (1984). 9 GOLDSTONE,A., KOENm, H., Lu, C. Y. Insulin stimulation of Ca 2+ fluxes and membrane transport processes in mouse kidney cortex is mediated by polyamine synthesis. [Abstract] Fed Proc 44, 1593 (1985). 10 GOTZSC~E,O. Decreased myocardial calcium uptake after isoproterenol in streptozotocin-induced diabetic rats. Lab Invest 48, 156-161 (1983). 1l GUARNtERt,C., FLAMmm,F., MUSCARI,C., CALDARERA,C. M. Involvement of calcium ions in the activation of ornithine decarboxylase by isoprenaline evaluated in situ in the perfused rat heart. BiochemJ 212, 241-243 (1983). 12 GUARNIERI,C., FLAMIGNI,F., MUSCARI,C., CALDARERA,C. M. O D e activity evaluated in perfused rat heart in response to catecholamines. In: Advances in Polyamine Research, Vol. 4, U. Bachrach, A. Kaye and R. Chayen (Eds), pp. 537 548, New York : Raven Press (1983). 13 HUNTER,n. R., HAWORTH, R. A., BERKOFF,H. A. Measurement of rapidly exchangeable cellular calcium in the perfused beating rat heart. Proc Nat Acad Sci USA 78, 5665 5668 ([980). 14 IQBAL,Z., KOENIG,H. Polyamine appear to be second messengers in mediating Ca 2+ fluxes and neurotransmitter release in potassium depolarized synaptosomes. Biochem Biophys Res Commun 133, 563 573 (1985). 15 KOENm, H., GOLDSTONE,A., Lu, C. Y. Testosterone induces a rapid stimulation ofendocytosis, amino acid and hexose transport in mouse kidney cortex. Biochem Biophys Res Commun 106, 346-353 (1982). 16 KOENIG,H., GOLDSTONE,A., Lu, C. Y. fi-Adrenergic stimulation of Ca 2+ fluxes, endocytosis, hexose transport and amino acid transport in mouse kidney cortex is mediated by polyamine synthesis. Proc Natl Acad Sci USA 80, 7210-7214 (1983). 17 KOENm,H., GOLDSTONE,A., Lu, C. Y. Pnlyamines regulate calcium fluxes in a rapid plasma membrane response. Nature 305, 530-534 (1983). 18 KOENm,H., GOLOSTONE,A. D. Lu, C. Y. Blood-brain barrier breakdown in brain edema following cold injury is mediated by microvascular polyamines. Bioehem Biophys Res Commun 116, 1039-1048 (1983).
Role of Polyamines in p-Adrenergic Effects
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19 KOENIG,H., FAN, C.-C., Lu, C. Y., GOLDSTONE,A. D. Polyamines as signals mediating enhanced heart contrac20 21 22 23 24
25 26 27 28 29 30 31 32 33 34 35 36
tility after androgenic and ~-adrenergic stimulation. [Abstract] J Neurochem 44[Suppl 1], $64 (1985). KOENIO,H., GOLDSTONE,A. D., Lu, C. Y., IQBAL, Z., FAN, C.-C., TROUT,J. J. Polyamines, hormone receptors and calcium fluxes. In: The Physiology ofPolyamines, U. Bachrach and Y. M. Heimer (Eds), Boca Raton, FL: CRC Press (1988, in press). LAU, C., SLOTKIN, T. A. Stimulation of rat heart ornithine decarboxylase by isoproterenol: evidence for posttranslational control of enzyme activity ? EurJ Pharmaco178, 99-105 (1982). LODOE,N.J., BASSETT,A. L., GELBAaD, H. The effect of high K + depolarization and verapamil on Ca 2+ fluxes in the canine myocardium. Pflfigers Arch 41}3, 222-224 (1985). LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L., RANDALL, R. J. Protein measurement with the Folin phenol reagent.J Biol Chem 193, 265-275 (1951). Lu, C. Y., GOLDSTONE,A. D., KOEmO, H. Various surface agonists induce a rapid Ca2+-dependent stimulation of endocytosis, hexose and amino acid transport in kidney cortex. [-Abstract] Fed Proc 42, 294 (1983). MARCH,J. D., BARRY,W. H., NEER, E. J., ALEXANDER,R. W., SMITH,T. W. Desensitization of chick embryo ventricles to the physiologic and biochemical effect of isoproterenol. Evidence for uncoupling of the fl-receptoradenylate cyclase complex. Circ Res 47, 493-501 (1980). METCALF,B. W., BEY, P., DANZlN, C., JUNO, i . J . , CASARA,P., VEVERT,J. P. Catalytic irreversible inhibition of mammalian ornithine decarboxylase (EC 4.1.1.17) by substrate and product analogues. J Am Chem Soc 100, 2551-2553 (1978). NAWATA,H., YAMAMOTO,R. S., POIRIER, L. A. Ornithine decarboxylase induction and polyamine levels in the kidney ofestradiol-treated castrated male rats. Life Sci 26, 689-698 (1980). REUTER,H. A. Localization offl-adrenergic receptors and effects ofnoradrenaline and cyclic nucleotides on action potentials, ionic currents and tension in mammalian cardiac muscle.J Physio1242, 429-451 (1974). REUTER,H. A. Properties of two inward membrane currents in the heart. Ann Rev Physio141,413 424 (1979). REUTER, H. A. Calcium channel modulation by neurotransmitters, enzymes and drugs. Nature 301, 569-374 (1983). RINALDI,M. L., CAPON'Z,J.-P., DEMAILLE,J. G. The cyclic AMP-dependent modulation of cardiac sarcolemmal slow calcium channels. J Mol Cell Cardio114, 279-289 (1982). STEINMAN,R. M., COHN, J. A. The interaction of soluble horseradish peroxidase with mouse peritoneal macrophages in vitro.J Cell Bio155, 18f~204 (1972). TABOR,C. W., TABOR, H. Polyamines. Ann Rev Biochem 53, 749 790 (1984). TSIEN,R. W. Cyclic AMP and contractile activity in heart. Adv Cyclic Nucleotide Res 8, 363-420 (1977). VAN BREEMEN, C., CASTEELS, R. The use of Ca-EGTA in measurements of *SCa effiux from smooth muscle. Pfliigers Arch 348, 239-245 (1974). WARNICA,J. W., A~TONY, P., GiBsoN, K., HARMS, P. The effect ofisoprenaline and propranolol on rat myocardial ornithine decarboxylase. Cardiovasc Res 9, 793-796 (1975).
The Role o f P o l y a m i n e s in ~-Adrenergic Stimulation o f Calcium Influx and M e m b r a n e T r a n s p o r t in Rat Heart Chien-Chung Fan and Harold Koenig*
Neurology Service, VA Lakeside Medical Center and the Department of Neurology, Northwestern University Medical School, Chicago, IL 60611, USA (Received 26 September 1986, acceptedin revisedform 6 May 1988) C.-C. FAN ANDH. KOI~NIG.The Role of Polyamines in//-Adrenergic Stimulation of Calcium Influx and Membrane Transport in Rat Heart. Journalof Molecularand CellularCardiology(1988) 20, 789--799. The/~-adrenergic agonist 1-isoproterenol induced an early (< 1 rain) stimulation of endocytosis, amino acid transport and hexose transport, monitored by the temperature-sensitive uptake of horseradish peroxidase, ~-aminoisobutyrate and 2-deoxyglucose, respectively, in rat ventricle cubes. This stimulation was time- and concentration-dependent and was maximum at 10-sM isoproterenol. The /~-adrenergic antagonist propranolol blocked isoproterenol stimulation of membrane transport, thereby confirming/Ladrenoceptor mediation; 2.5 mM EGTA, 1 mM LaC12 and 100 ,UMverapamil blocked the hormonal response without affecting basal transport. The calcium ionophore A23187 caused an acute stimulation of endocytosis, hexose and amino acid transport. Isoproterenol rapidly (< 30 s) stimulated 45Ca2 + influx. These data suggest that stimulus-response (stimulus-"transport") coupling is meditated by a rise in cytosolic Ca 2+ concentration. A rapid ( < 30 to 60 s) increase in ornithine decarboxylase (ODC) activity, followed by an early ( < 1 to 2 min), sustained increase in putrescine, spermidine and spermine concentrations was evoked by 10-7M isoproterenol. The ODC inhibitor ~-difluoromethylornithine (DFMO, 5 mM) suppressed the isoproterenol-induced increase in ODC and polyamine levels and the stimulation of 45Ca influx, endocytosis, hexose transport, and amino acid transport. Putrescine (0.5 m~) negated DFMO inhibition and restored the increase in polyamines, '~SCa influx, endocytosis, and transport of hexose and amino acid. These data suggest that polyamine synthesis is involved in isoproterenol stimulation of Ca 2+ influx and membrane transport functions in ventricular myocardium. These findings are consistent with a model for signal transduction and stimulu~response coupling in which polyamines function as intracellular messengers to generate cytosolic Ca 2+ signals by stimulating Ca 2 + influx. KEy WOROS: Isoproterenol; /~-Adrenergic agonist; /~-Adrenoceptor; Ornithine decarboxylase; Polyamines; Putrescine; Spermidine; Spermine; Ca2+influx; Membrane transport; Endocytosis; Amino acid transport; Hexose transport; Signal transduction.
Introduction /~-Adrenergic modulation of voltage-sensitive c a l c i u m c h a n n e l s in m y o c a r d i a l cells initiates a slow i n w a r d c u r r e n t c a r r i e d m a i n l y b y C a z + t h a t p r o f o u n d l y influences c a r d i a c f u n c tion a n d constitutes the m o s t i m p o r t a n t neuroregulatory control over heart action [29]. C a c h a n n e l m o d u l a t i o n a n d i n c r e a s e d C a influx a r e t h o u g h t to be m e d i a t e d b y a n e l e v a t i o n o f i n t r a c e l l u l a r c A M P [34] a n d p r o t e i n p h o s p h o r y l a t i o n [ 3 / ] , l e a d i n g to a n i n c r e a s e in the a v e r a g e n u m b e r o f f u n c t i o n a l C a c h a n n e l s p e r cell, a n d also c h a n g e s in the t i m e course o f b o t h a c t i v a t i o n a n d i n a c -
t i v a t i o n o f C a c h a n n e l s [-2, 30]. H o w e v e r , the m o l e c u l a r m e c h a n i s m s u n d e r l y i n g the c o n t r o l o f C a c h a n n e l a c t i v i t y in c a r d i a c muscle cells are still u n e l u c i d a t e d . R e c e n t studies in o u r laboratory have shown that a transient increase in o r n i t h i n e d e c a r b o x y l a s e ( O D C ) , the first a n d r a t e - l i m i t i n g e n z y m e o f the p o l y a m i n e s y n t h e t i c p a t h w a y , a n d a rise in polya m i n e levels a r e e a r l y c e l l u l a r events in m o u s e kidney cortex following receptor activation by t h e / / - a d r e n e r g i c a g o n i s t 1-isoproterenol [-16], a s well as b y testosterone [17], t r i i o d o t h y r o n i n e [-8], a n d insulin [9]. T h i s p o l y a m i n e synthesis has b e e n f o u n d to be essential for
* Please send reprint request to : Professor Harold Koenig, Neurology Service, VA Lakeside Medical Center, 333 E. Huron Street, Chicago, IL 60611, USA. 0022-2828/88/090789 + 11 $03.00/0
9 1988 Academic Press Limited
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hormonal stimulation of Ca z+ fluxes and several Ca 2 +-dependent membrane transport processes (reviewed in [20]). We report here experiments suggesting that isoproterenol stimulation of Ca z + fluxes and Ca 2 +dependent membrane transport processes in rat heart appears to be mediated by rapid polyamine synthesis. A brief account of some of these results has appeared in abstract form
[53. Methods
Incubations Female Sprague-Dawley rats ( ~ 150 to 200 g) were used for these studies. Animals were killed by decapitation and hearts were rapidly excised and placed in an ice-cold physiological salt solution (PSS) containing (in raM) : Na 122, KC1 5, CaC12 2.7, MgSO4 1.2, N a z H P O , ~ 0.6, glucose 6, H E P E S buffer (pH 7.4) 39. Ventricles were dissected free and cut into .-~0.5 m m cubes with a McIlwain tissue chopper. Incubations were carried out in 25-ml plastic beakers in PSS with appropriate additions at 37~ under 95% O z in a Dubnoff shaking incubator.
Rates of endocytosis, hexose and amino acid transport Endocytosis, amino acid transport and hexose transport were measured by monitoring the temperature-sensitive uptake of horseradish peroxidase (HRP), e-[ 1-x4C]aminoiso butyrate (AIB), and 2-deoxy-D-[1-aH]-glu cose (DG) in heart cubes as previously described for kidney slices [7, 15]. H R P (1 mg/ml), 14C-AIB (0.1mM, 4/~Ci/ml) and 3H-DG (0.1 raM, 1 #Ci/ml) were added to the incubation medium. Incubations were terminated by placing beakers on ice and washing ventricle cubes three times with fresh medium by centrifugation. Corrections for nonspecific uptake were made by subtracting the amount of tracers taken up during parallel incubations at 0~ Slices were sonicated in H 2 0 for measurement of protein, H R P and radioactivity (by scintillation spectrometry).
Rate of 45Ca influx 45Ca influx was measured by a modification of the method of Lodge et al. [22]. Heart
cubes were pre-incubated briefly (5 min) in PSS with appropriate additions, transferred to fresh media containing 45Ca (1 to 2 g C i / ml) and isoproterenol or vehicle, and incubated with constant shaking under 95% 0 2 - 5 % CO2 at 37~ for 30, 60 and 90s. Incubations were terminated by chilling on ice, and aliquots of incubates were washed twice in 3 vols of ice-cold PSS by centrifugation. The pelleted heart cubes were resuspended in PSS containing 7.5mM Ca z+ and 6 m~ EGTA, and shaken for 1 h at 0~ in order to remove most of the extracellular 45Ca2+. To correct for residual extracellular 45Ca, heart cubes were pre-incubated and incubated at 0~ as described above, and subjected to the same wash procedure. The 0~ uptakes were then subtracted from the 37~ uptakes. In principle, the exchange and removal of extracellular free and bound 45Ca2+ should be facilitated by the large Ca 2 + buffering pool provided in this wash solution, while cooling to 0~ will decrease transmembrane Ca 2 + movement without greatly impeding the diffusion of extracellular Ca, and thus preserve much of the intracellular 45Ca [1, 22, 35]. Cells injured during sectioning are believed to become isolated owing to a reduction in intercellular junction permeability induced by the membrane lesion [4], and the C a - E G T A wash procedure should deplete such damaged cells of 45Ca. In the present experiments 0~ uptake was timedependent and insensitive to additions, and was generally 25 to 45% of the 37~ uptake in basal or DEMO-inhibited heart, and 15 to 22% of that in isoproterenol-stimulated heart. The initial rates of 45Ca influx in unstimulated heart cubes were comparable to these obtained by Lodge et al. [22] in thin canine ventricle sheets, and in the isolated, perfused rat heart [10, 13].
Analytical methods Protein was determined according to Lowry et al. [23], using bovine serum albumen as standard. H R P was assayed according to Steinman and Cohn [32]. Polyamines were extracted by homogenizing tissue samples in cold 0.2 ~ perchloric acid, and removing the insoluble residue by centrifugation. Polyamines were measured by spectrophotofluori-
Role of Polyamlnes in p-Adrenergic Effects
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FIGURE 1. Effect ofisoproterenol on the time course of (a) HRP, (b) DG and (c) A1B uptake by rat heart in vitro. Ventricular cubes were pre-incubated in PSS for 10 rain at 37~ At zero time the medium was replaced by fresh medium coataining HRP (1 mg/ml), [ 14 C]AIB (0.1 mM, 4#Ci/ml), [ 3 H]DG (0.1 raM, 1/~Ci/ml), and 10- ? Misoproterenol (O . . . . C)) or no hormone ( 0 - 0 ) . Tissue samples were removed for study at each time point of incubation commencing at 0.5 rain. Data were corrected for nonspecific uptake as described in Methods. Results are means _ S.E.M.(n = 3). * P < 0.05 (Student's t-test). m e t r y o f the d a n s y l a t e d derivatives after separation by thin-layer chromatography [27]. Recoveries of polyamines, as assessed with a n i n t e r n a l s t a n d a r d , a v e r a g e d 70%, a n d d a t a were corrected for these recoveries. O r n i t h i n e d e c a r b o x y l a s e activity was determ i n e d b y a modification [17] of the m e t h o d o f D j u r h u u s [3]. Tissue samples were h o m o g e nized in a p p r o x i m a t e l y 5 vols of 50mM s o d i u m - p h o s p a t e buffer, p H 7.2, c o n t a i n i n g 5 mM dithiothreitol, 0.1 m ~ e t h y l e n e d i a m i n e
tetraacetic acid ( E D T A ) a n d 40 mM p y r i d o x a l phosphate. H o m o g e n a t e s were centrifuged at 12000 • g for 20 min at 4~ a n d the pellets were discarded. S u p e r n a t a n t (0.1ml) was a d d e d to 0.1 ml o f i n c u b a t i o n m e d i u m consisting of 50mM s o d i u m - p h o s p h a t e buffer, p H 7.2, 5ram dithiothreitol, 200#M-pyridoxal phosphate, 0.1 mM L-ornithine, a n d 0.3/~Ci of 3 - 3 H ( n ) - o r n i t h i n e (20 C i / m m o l e -1, New E n g l a n d Nuclear). T h e tubes were i n c u b a t e d for 2 h at 37~ and then were cooled to 4~
792
C.-C. Fan and H. Koenig
Incubation mixture (40/A) was applied to Whatman P-81 cation exchange paper, and paper strips were subjected to descending chromatography for 3 h with 0.1 M N H 4 O H to elute unreacted ornithine. The strips were dried and the spots corresponding to a putrescine standard detected by ninhydrin were cut out, placed in scintillation vials with 1 ml of deionized water, and heated at 50~ for 2 h. Aquassure scintillation fluid (10ml) (New England Nuclear) was added to each vial and the radioactivity was counted in a Beckman LS-250 liquid scintillation counter. Enzyme controls containing the complete assay mixture and boiled enzyme were run with each determination and the values were subtracted from the experimental values. O D e activity was expressed as pmol of putrescine H - 1. O D e activity was linear with time and protein in the range in which determinations were made. All results are presented as means _ standard errors. The statistical significance of differences between means was evaluated by Student's t-test.
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HRP, AIB and DG uptake The temperature-dependent uptake of H R P was linear between 0 and 10 min, and between 10 and 60 min, with the rate of uptake being two-fold greater during the first 10 min than during the subsequent 10 to 60
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3H-DG was purchased from Amersham (Arlington Heights, IL). L-[3-3H(n)]orni thine, 14C-AIB, 4Sea, and Aquasol came from New England Nuclear (Boston, MA). Horseradish peroxidase (Type II) and 1isoproternol came from Sigma Chemical Co. (St. Louis, MO). 0~-Difluoro-methylornithine was supplied by Merrell Dow Research Center (Cincinnati, OH), A23187 was obtained from Calbiochem-Behring (LaJolla, CA) and verapamil Dx-isopropyl-D-(Nmethyl-N-homoveratryl)-c<-amino-propyl-3, 4-dimethoxyphenylacetonitrile hydrochloride] from Knoll Pharmaceutical Co. (Whippany, NJ). Female albino SpragueDawley rats were from Holtzman (Madison, WI).
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F I G U R E 2. Effect of isoproterenol concentration on (a) HRP, (b) AIB and (c) DG uptake by rat heart. After a 10 min pre-incubation in PSS at 37~ ventricular cubes were placed in fresh medium containing HRP, [14C]AIB and [SH]DG and various concentrations of isoproterenol and incubated for 5 min at 37~ Hormonefree control uptake values were: HRP, 4 5 _ 3.7 ng/5 m i n / m g protein; DG, 4 1 3 + 3 1 . 5 pmol/5 min/mg protein; AIB, 151 + 19.4 pmol/5 min/mg protein. Additional details are given in Methods. Results are means + s.~.M. (n = 3). *, **, ***: P < 0.05, 0.01 and 0.0(31 (vs. control).
min (Fig. 1). This uptake varied linearly with solute concentration (1 to 10 mg/ml) consistent with fluid volume endocytosis. Isoproterenol increased the rate of H R P uptake by 50 to 120% at 5 min. This increase developed immediately and was readily detected at 1 min when a greater concentration of H R P (25 mg/ml) was used. Stimulation of H R P uptake varied with isoproterenol concentration and was greatest at 10 -8 M (Fig. 2). The temperature-sensitive uptake of AIB and DG by heart cubes was linear for about 2 min and then gradually declined over the duration of the incubation (Fig. 1). Isoproterenol increased t h e rate of AIB and DG
Role of Polyamines in ~-Adrenergic Effects 8O
793
2.5 c
2.0-
o Q.
4O
L5
0
=~ 0 . 5 3
2oo l l(b)
0 0
i [ 30 60 Incubation time (s)
i 90
FIGURE 4. Effects ofisoproterenol on 4SCa influx in rat heart. Ventricular cubes were incubated at 37~ in PSS containing 4Sea without (O O) and with (Q) - - - Q) ) 10- v Misoproterenol. Results are means _+ S.E.M.(n = 3).
i 100 c
o
200
I00
0
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w
o
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FIGURE 3. Effect of E G T A , lanthanum, vcrapamil and A23187 on basal and isoprotcrcnol-stimulatcd
uptake of (a) HRP, (b) AIB and (c) DG by rat heart. Ventricular cubes were pre-ineubated at 37~ for 10 min in 2.5mM EGTA in Ca2+-free PSS, 1 mM lanthanum chloride (La3+), 100mM verapamil (VER), or [0•M A23187 in PSS containing 2.7mM Ca 2+, and then were incubated for 5 min in the same medium containing HRP, [14C]AIB, [3H]DG, 10-TM isoproterenol (m) or no hormone ([-]). Data are means _ S.E.M.(n = 3).
u p t a k e b y 40 to 100% at 5 min. Isoproterenol s t i m u l a t i o n o f A I B a n d D G u p t a k e was c o n c e n t r a t i o n - d e p e n d e n t a n d m a x i m u m at 10-SM (Fig. 2). The /Ladrenergic antagonist propranolol (10-TM) blocked the increase in H R P , A I B
a n d D G u p t a k e i n d u c e d b y 10-SM isoproterenol, b u t h a d no effect on basal u p t a k e of these substrates ( d a t a not shown). This confirms t h a t isoproterenol s t i m u l a t i o n of these m e m b r a n e t r a n s p o r t processes is m e d i a t e d b y ~ - a d r e n o r e c e p t o r activation. Figure 3 shows the effect o f E G T A , L a 3+, v e r a p a m i l a n d A23187 on basal a n d isoproterenol-stimulated H R P , A I B a n d D G uptake. T h e C a 2+ c h e l a t o r E G T A (2.5mM, no a d d e d C a 2+) and the C a antagonist L a 3 + (1 mM) caused a statistically insignificant decrease in b a s a l H R P , A I B a n d D G u p t a k e and abolished isoproterenol stimulated u p t a k e of these substrates into ventricle cubes. T h e Ca c h a n n e l blocker v e r a p a m i l (100/~M) in the presence of 2.7 mM C a 2+ did not alter basal u p t a k e o f H R P , A I B a n d D G , b u t abolished the isoproterenol response. I n contrast, the Ca i o n o p h o r e A23187 (10#M) i n d u c e d a two-fold increase in the u p t a k e of H R P , A I B a n d D G , a n d a d d i t i o n o f isoproterenol (10-SM) caused no further increase in this uptake. These experiments indicate that isoproterenol stimulation of endocytosis, hexose a n d a m i n o acid t r a n s p o r t requires extracellular Ca 2 +, C a 2 + b i n d i n g to extracellular sites, a n d Ca 2+ t r a n s p o r t in heart, as it does in kidney cortex [7].
"Sea Influx Isoproterenol (10-TM) i n d u c e d substantial increases (81 to 162%) in 45Ca into ventricle
794
C.-C. Fan and H. Koenlg
(a) 300
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~- * * %
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200
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(b)
**
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bation in the continued presence of the agonist [Fig. 5(a)]. All three polyamines rapidly increased to m a x i m u m levels at 5 min, and remained elevated during a 60 min incubation (Fig. 5). The rank order of increase of these constituents (percentage of control value) after 1 min of incubation with isoproterenol was: O D C (246%, P < 0.001) > putrescine (184%, P < 0.01) > spermidine (182%, P < 0.05) > spermine (128%, P > 0.05 < 0.1). Propranolol (10-6M) blocked the isoproterenol (10 -v~)-induced increase in O D C and polyamine levels in ventricle slices (data not shown). These data show that an increase in O D C activity and enhanced polyamine synthesis are very early /7-adrenoceptor-mediated events in isoproterenol-stimulated rat heart.
Role of polyamines in membrane transport
(c)
e-Difluoromethylornithine (DFMO), a specific, irreversible, enzyme-activated inhibitor of O D C [26, 33], was used to assess the role of I00 polyamines in isoproterenol stimulation of il I [ I ii 1 m e m b r a n e transport functions. Figure 6 01 2 5 Io 60 shows that 5mM D F M O abrogated the Time of ineub0tion (rain) increase in endocytosis, hexose transport and FIGURE 5. lsoproterenol induces an acute increase amino acid transport induced by isoproin ODC activity and polyamine lavels in rat heart. Ventricular cubeswere incubated at 37~ with 10-v~ isopro- terenol. Putrescine (0.5m~), the product of terenol for various times and assayed for ODC and O D C activity, reversed the effect of D F M O polyamines as described in Methods. Zero-time control and restored the increase in endocytosis, values were: (a) ODC (O), 971-t-163 pmol/h/mg hexose transport and amino acid transport. protein; putrescine (C)), 0.143 -t- 0.026 nmol/mg protein; (b) spermidine, 0.66-I-0.14 mg protein; (c) D F M O blocked the isoproterenol-induced spermine, 1.93 -I-0.16 nmol/mg protein. Data are means increase in polyamine levels, and putrescine 4- S.E.M.(n = 3--4). repleted heart polyamines (Table 1). D F M O also inhibited basal membrane transport (not cubes during 30 to 90s incubations (Figs 4 shown) and decreased polyamine concentrations during a 15 min incubation (Table 1). and 7). Putrescine mimicked the effect of isoproterenol in that it stimulated membrane transODC activity andpolyamine port (not shown) and increased the levels of concentrations polyamines in rat heart during a 15 min incuIsoproterenol, 50 nmol/kg body wt intraperibation (Table 1). These results provide pertoneally, evoked a rapid ( < 2 rain) increase in suasive evidence for the view that rapid the concentrations of heart polyamines in vivo polyamine synthesis is involved in the acute (data not shown). Isoproterenol also induced effect of isoproterenol on membrane transport an early stimulation of O D C activity and an in rat heart. accumulation of polyamines in ventricle cubes in vitro (Fig. 5). O D C activity increased Role of polyamines in Ca z + influx rapidly after 10-TM isoproterenol to a maximum at 1 to 2 min and then declined but D F M O was also used to evaluate the role of still remained elevated after 60 min of incu- polyamines in isoproterenol stimulation of 200
%
%
%
Role of Polyamines in [i-Adrenergic Effects
200 -
o
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c u
((I + +
150
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+
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+
+
0
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0
~-
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FIGURE 6. DFMO blocks isoproterenol stimulation of endocytosis, amino acid transport and hexose transport in rat heart, and putrescine negates the DFMO effect. Ventricular cubes were pre-incubated for 10 min at 37~ in PSS with 5 mM DFMO, DFMO and 0.5 mM putrescine (PUT), or no additions. Cubes were transferred to fresh medium of the same composition containing HRP, [14C]AIB, [3H]DG, and 10- 7 ~ isoproterenol (ISO), or no hormone (control, C) and incubated for 5 min. Data are means ___S.E.M. (n = 3). Control uptake values (means +_ S.E.M.) were: (a) HRP, 29.3 _+ 2.8 ng/5 min/mg protein; (b) AIB, 142 -I- 18 pmol/5 min/mg protein; (c) DG, 216 -t- 23 pmol/5 rain/rag protein. *,**, P < 0.05, 0.01, (vs. control). ~', ~;~,P < 0.05, 0.01, (vs. isoproterenol).
Ca z + influx. DFMO blocked the stimulation of 4SCa influx evoked by isoproterenol during i n c u b a t i o n s o f 30 to 90 s (Fig. 7). P u t r e s c i n e n e g a t e d D F M O i n h i b i t i o n a n d p a r t i a l l y res t o r e d t h e i n c r e a s e i n 4 5 C a influx. D F M O d i d n o t a f f e c t 4 5 C a i n f l u x d u r i n g a 6.5 m i n i n c u bation; however, putrescine enhanced 45Ca i n f l u x d u r i n g a 6.5 m i n i n c u b a t i o n . T h e s e data support the inference that rapid polya m i n e s y n t h e s i s is i n v o l v e d i n i s o p r o t e r e n o l -
mediated heart.
s t i m u l a t i o n o f C a 2+ i n f l u x i n r a t
Discussion T h e d a t a p r e s e n t e d i n t h e first p a r t o f this paper have demonstrated that nanomolar concentrations ofisoproterenol induce a rapid (1-2 min) stimulation of endocytosis, amino
T A B L E 1. Effect of D F M O a n d putrescine on the rapid increase in heart polyamines induced by isoproterenol
Treatment No addition 10-7M isoproterenol + 5mM D F M O + 5 mM D F M O + 0.5 mM putrescine 5 mM D F M O 0.5 mM putrescine
Putrescine ( n m o l / m g protein)
Spermidine ( n m o l / m g protein)
Spermine ( n m o l / m g protein)
0.123 -I- 0.001 0.196 -I- 0.013 r 0.110 -t- 0.010 d
0.677 _-/-0.036 1.144 + 0.060 c 0.460 -I- 0.064 d
1.256 -I- 0.053 2.037 -I- 0.156 ~ 0.829 ___0.103 d
0.154 -t- 0.016 e 0.096 -I- 0.012" 0.120 _ 0.013
0.900 -t- 0.026 ~ 0.509 -I- 0.066 a 0.849 _ 0.057 a
1.627 -t- 0.023 f 0.883 -t- 0.056 b 1.596 _ 0.106 a
Heart slices were pre-incubated at 37~ for 10 min in PSS with 5 mM DFMO, 0.5 mM putrescine, DFMO and 0.5ram putrescine, or no addition, and then were incubated for 5 rain with or without 10-TM isoproterenol. Corrections for nonspecific uptake of putrescine were made by subtracting the 0~ uptake. Results are means __S.E.M. (n = 3). a,b,r p < 0.05, 0.01, 0.001 (vs. no addition), d: p < 0.001 (vs. isoproterenol). ~,f,g: P < 0.05, 0.01, 0.001 (vs. isoproterenol and DFMO).
796
C.-C. Fan and H. Koenig
2.5 -
2.0
]
Control
~]
[]
ISO
[[]-] Putrescine
[]
ISO +DFMO
DFMO
ISO + DFMO+putrescine
o.
-6 E c
T
1.5
+
:;
+ + +
g~ 0.5
30s
60 s
90 s
FIGURE 7. DFMO abolishes the isoproterenol-induced increment in '*SCa influx in rat heart, and putrescine negates DFMO inhibition. Ventricular cubes were preincubated for 5 min at 37~ with 10ram DFMO, DFMO and 1mM putrescine, 1 mM putrescine, or no agent, transferred to fresh medium of the same composition containing 4SCa and 10-vM isoproterenol or no hormone (control), and incubated for the given time periods. Data are means + s.E.~t. (n=3). **: P<0.01 (vs. control) ",~, "~r162 P<0.05, 0.01 (vs. isoproterenol); +, + + : P<0.05, 0.01 (vs. isoproterenol + DFmo). acid transport and hexose transport in rat ventricular myocardium. This m e m b r a n e transport response is mediated by fladrenoreceptor occupancy as it is elicited by the specific fl-adrenergic agonist isoproterenol, demonstrates h o r m o n e concentration dependence, and is abrogated by the fladrenergic antagonist propranolol. These experiments further show that isoproterenol stimulation of m e m b r a n e transport requires the presence of Ca 2+ in the extracellular medium, and appears to involve an increased inflow of 4 5 Ca 2 + . Enhanced Ca 2 + influx by isoproterenol has been previously reported during depolarization [28] and in the perfused beating heart [10, 13]. O u r results for basal 4~Ca uptake in rat heart cubes are in good agreement with those obtained in isolated perfused rat heart [10, 13~, and in thin canine heart sheets [22], when their results are expressed as Ca z+ uptake per min per mg protein. I n the work of H u n t e r et al. [13] and Gotzsche [10], the relative increase after isoproterenol stimulation was 46% and 39 to 53% at 1.5 x 10 -6 and 10-4M, as compared with 8 1 - 1 6 2 % at 10-7M isoproterenol in our
experiments. E n h a n c e d Ca 2+ influx appears to be important for the isoproterenol-induced m e m b r a n e transport response as it was abolished by the Ca 2 + chelator E G T A , the Ca 2 + antagonist La 3 +, and the organic Ca 2 + transport blocker verapamil. These data support the view that fl-adrenergic stimulation of cardiac myocytes increases cytosolic Ca 2+ concentration by enhancing the inflow of extracellular Ca 2+ across the sarcolemma. It is evident from these results that the rates of endocytosis, hexose transport and amino acid transport in ventricular m y o c a r d i u m are modulated by fl-adrenergic stimulation in a coordinate manner. These experiments have established that isoproterenol causes a rapid, fl-adrenoceptormediated stimulation of O D C activity and polyamine synthesis in rat ventricular myocardium. T h e early increase in heart O D C and polyamine levels is similar to that evoked by isoproterenol in mouse kidney cortex [16]. Previous studies have indicated that fladrenergic agonists increase heart O D C activity after a lag period of 1 to 4 h [6, 21, 36]. Synthesis of prostaglandins and cyclic A M P
Role of Polyamlnes in ~-Adrenerglc Effects
appears to be involved in the rapid stimulation of ODC activity and membrane transport functions by isoproterenol, as these responses are blocked by dexamethasone, aspirin, and 2',5'-dideoxyadenosine, inhibitors of arachidonate release, cyclooxygenase, and adenylate cyclase respectively, in mouse kidney cortex ([16, 20]; H. Koenig, A. D. Goldstone, C. Y. Lu, unpublished observations). In addition the prostaglandins PGA2 and PGE2, and dibutyryl-cyclic AMP have been shown to mimic the effects of isoproterenol in stimulating ODC activity and membrane transport processes [24]; H. Koenig, A. D. Goldstone, C.Y. Lu, unpublished observations). We have therefore suggested that this rapid stimulation of ODC activity likely represents a fl-receptor-mediated, Ca z +-, prostaglandin-, and cyclic AMPdependent activation of a latent ODC via a posttranslational process, possibly a rapidly reversible phosphorylation-dephosphorylation sequence involving O D C or an ODC regulating protein [16, 17, 20]. In addition, cytoskeletal participation appears to be important for isoproterenol stimulation of ODC activity and membrane transport functions as this stimulation is blocked by the microfilament-disrupting agent cytochalasin B and the microtubule-disrupting agent colchicine [20]; H. Koenig, A. D. Goldstone & C. Y. Lu, unpublished data). The timedependent decline in heart ODC activity in the continued presence of isoproterenol could reflect a desensitization effect resulting from uncoupling of the fl-receptor-adenylate cyclase complex [25"]. Guarnieri et al. [11, 12] have described an early fl-adrenoceptor-mediated increase in ODC in isolated, perfused rat heart which manifests Ca 2 +- and calmodulindependence and is independent of protein synthesis. In the present study the rank order of increase of polyamines at 1 rain was putrescine > spermidine > spermine. This rank order is consistent with the precursorproduct relationship observed in the ODCmediated pathway for polyamine synthesis [33]. Direct support for this inference comes from the finding that DFMO, a specific, irreversible, enzyme-activated, suicide inhibitor of ODC [26, 33], blocked the rapid isoproterenol-induced increase in polyamine
797
levels. Although the kinetic properties of ODC were not characterized, the observed ODC activity in isoproterenol-stimulated heart, ~-,30 pmol/min/mg protein, would appear to be too low to account for the observed increment in total polyamines, ,-, 145 pmol/min/mg protein. A similar disparity between ODC product and ODC activity is apparent in other rapid responses, including testosterone-J17] and isoproterenolstimulated kidney cortex [16"j, cryoinjured brain [18], and potassium depolarized brain synaptosomes [14]. Although the basis for this disparity is uncertain, its consistent occurrence in a variety of rapid cell responses to external stimuli strongly suggests that the catalytic activity of ODC in intact stimulated cells may be much greater than that measured in vitro in broken cell preparations or cytosolic extracts. These experiments provide direct evidence that rapid polyamine synthesis is involved in the isoproterenol-mediated increase in Ca 2+ influx and Ca 2 +-dependent membrane transport processes in ventricular myocardium, as it is in kidney cortex [16]. Thus D F M O suppressed isoproterenol stimulated polyamine synthesis and concurrently blocked the increase in 45Ca2 + influx, endocytosis, amino acid transport and hexose transport. Repletion of heart polyamines with putrescine negated D F M O inhibition and restored the increment in 4SCa2+ influx and Ca 2+d e p e n d e n t membrane transport functions. These data support the hypothesis that polyamines function as intracellular messengers to augment Ca 2 + in-flow across the sarcolemma. The consequent elevation in sarcoplasmic C a 2 + concentration would trigger the coordinate stimulation of endocytosis, hexose transport and amino acid transport and stimulate other Ca 2 +- (or Ca 2 +-calmodulin-) and polyamine-dependent reactions, e.g. excitation-cotraction coupling, in cardiac myocytes. Thus a recent study has implicated polyamines in the fl-adrenergic stimulation of heart contractility and rate [19]. The data described in this paper support the hypothesis that rapid ODC activation and polyamine synthesis' play a role in signal transduction and transmission and stimulusresponse coupling in heart cells. In this model transduction of the fl-adrenergic signal into
798
C.-C. Fan and H. K o e n i g
an intracellular message for the generation of a Ca 2 + signal involves a receptor- and Ca 2 +dependent activation of a latent O D C located in or near the sarcolemma. Transmission of the/~-adrenergic message is effected by polyamines initially synthesized in the subsarcolemmal microdomain. Newly synthesized polyamines may serve as messengers to generate Ca/+ signals by regulating Ca channel activity, Na/Ca exchange, and Ca 2 + influx by one or more of the following effects: (1) by displacing bound calcium via a cation exchange reaction; (2) by binding to anionic sites and acting as counterions to decrease surface charge and potential; (3) by modulating the phosphorylation states of proteins associated with the calcium channel or
calcium exchanger through polyaminedependent kinases or phosphatases. In this model the signaling action of polyamines is terminated when they bind to anionic sites or are degraded. It remains for future studies to elucidate the precise molecular mechanisms by which polyamines regulate transmembrane Ca 2 + movements.
Acknowledgements We thank Dr A. D. Goldstone and Mr C. Y. Lu for valuable assistance in several experiments, and Mrs T. Howell for the secretarial work. The research was supported by the Veterans Administration Research Service and N I H grants NS 18047 and H L 26835.
References 1 AARONSON,P., VANBREEMEN,R., LOUTZENHISER,R., KOLBER, M. A. A new mcthod for measuring the kinetics of transmembrane calcium-45 efflux in the smooth muscle of the guinea pig taenia coli. Life Sci 25, 1781 1790 (1979). 2 BEAN,B. P., Nowvmv, M. C., TSXEN,R. W. fl-Adrenergic modulation of calcium channels in frog ventricular heart cells. Nature 307, 371-375 (1984). 3 DJURHUUS,R. Ornithine decarboxylase assay based upon the retention of putrescine by a strong cation-exchange paper. Anal Biochem 113, 352-355 (1981). 4 DE MELLO,W. C. Modulation ofjunctional permeability. Fed Proc 43, 2692 2696 (1984). 5 FAN,C.-C., KOEmO, H. fl-Adrenergic stimulation of calcium fluxes and membrane transport processes in rat heart is mediated by polyamines. [Abstract] Fed Proc 43, 735 (1984). 6 FULLER,R. W., HEMRICK,S. K. Studies of noradrenergic influence on cardiac ornithine decarboxylase in rats. J Mol Cell Cardiol 10, 1031 1036 (1978). 7 GOLDSTONE,A. D., KOENIO,H., Lu, C. Y., TROUT,J. J. fi-Adrenergic stimulation evokes a rapid Ca 2+-dependent stimulation of endocytosis, hexose and amino acid transport associated with increased Ca 2+ fluxes in mouse kidney cortex. Biochem Biophys Res Commun 114, 913 921 (1983). 8 GOLDSTONE,A., KoEm% H., Lu, C. Y. Triiodothyronine (Ts) stimulation of Ca 2+ influx and membrane transport processes in mouse kidney cortex is mediated by polyamine synthesis. [-Abstract] Fed Proc 43, 735 (1984). 9 GOLDSTONE,A., KOENm, H., Lu, C. Y. Insulin stimulation of Ca 2+ fluxes and membrane transport processes in mouse kidney cortex is mediated by polyamine synthesis. [Abstract] Fed Proc 44, 1593 (1985). 10 GOTZSC~E,O. Decreased myocardial calcium uptake after isoproterenol in streptozotocin-induced diabetic rats. Lab Invest 48, 156-161 (1983). 1l GUARNtERt,C., FLAMmm,F., MUSCARI,C., CALDARERA,C. M. Involvement of calcium ions in the activation of ornithine decarboxylase by isoprenaline evaluated in situ in the perfused rat heart. BiochemJ 212, 241-243 (1983). 12 GUARNIERI,C., FLAMIGNI,F., MUSCARI,C., CALDARERA,C. M. O D e activity evaluated in perfused rat heart in response to catecholamines. In: Advances in Polyamine Research, Vol. 4, U. Bachrach, A. Kaye and R. Chayen (Eds), pp. 537 548, New York : Raven Press (1983). 13 HUNTER,n. R., HAWORTH, R. A., BERKOFF,H. A. Measurement of rapidly exchangeable cellular calcium in the perfused beating rat heart. Proc Nat Acad Sci USA 78, 5665 5668 ([980). 14 IQBAL,Z., KOENIG,H. Polyamine appear to be second messengers in mediating Ca 2+ fluxes and neurotransmitter release in potassium depolarized synaptosomes. Biochem Biophys Res Commun 133, 563 573 (1985). 15 KOENm, H., GOLDSTONE,A., Lu, C. Y. Testosterone induces a rapid stimulation ofendocytosis, amino acid and hexose transport in mouse kidney cortex. Biochem Biophys Res Commun 106, 346-353 (1982). 16 KOENIG,H., GOLDSTONE,A., Lu, C. Y. fi-Adrenergic stimulation of Ca 2+ fluxes, endocytosis, hexose transport and amino acid transport in mouse kidney cortex is mediated by polyamine synthesis. Proc Natl Acad Sci USA 80, 7210-7214 (1983). 17 KOENm,H., GOLDSTONE,A., Lu, C. Y. Pnlyamines regulate calcium fluxes in a rapid plasma membrane response. Nature 305, 530-534 (1983). 18 KOENm,H., GOLOSTONE,A. D. Lu, C. Y. Blood-brain barrier breakdown in brain edema following cold injury is mediated by microvascular polyamines. Bioehem Biophys Res Commun 116, 1039-1048 (1983).
Role of Polyamines in p-Adrenergic Effects
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19 KOENIG,H., FAN, C.-C., Lu, C. Y., GOLDSTONE,A. D. Polyamines as signals mediating enhanced heart contrac20 21 22 23 24
25 26 27 28 29 30 31 32 33 34 35 36
tility after androgenic and ~-adrenergic stimulation. [Abstract] J Neurochem 44[Suppl 1], $64 (1985). KOENIO,H., GOLDSTONE,A. D., Lu, C. Y., IQBAL, Z., FAN, C.-C., TROUT,J. J. Polyamines, hormone receptors and calcium fluxes. In: The Physiology ofPolyamines, U. Bachrach and Y. M. Heimer (Eds), Boca Raton, FL: CRC Press (1988, in press). LAU, C., SLOTKIN, T. A. Stimulation of rat heart ornithine decarboxylase by isoproterenol: evidence for posttranslational control of enzyme activity ? EurJ Pharmaco178, 99-105 (1982). LODOE,N.J., BASSETT,A. L., GELBAaD, H. The effect of high K + depolarization and verapamil on Ca 2+ fluxes in the canine myocardium. Pflfigers Arch 41}3, 222-224 (1985). LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L., RANDALL, R. J. Protein measurement with the Folin phenol reagent.J Biol Chem 193, 265-275 (1951). Lu, C. Y., GOLDSTONE,A. D., KOEmO, H. Various surface agonists induce a rapid Ca2+-dependent stimulation of endocytosis, hexose and amino acid transport in kidney cortex. [-Abstract] Fed Proc 42, 294 (1983). MARCH,J. D., BARRY,W. H., NEER, E. J., ALEXANDER,R. W., SMITH,T. W. Desensitization of chick embryo ventricles to the physiologic and biochemical effect of isoproterenol. Evidence for uncoupling of the fl-receptoradenylate cyclase complex. Circ Res 47, 493-501 (1980). METCALF,B. W., BEY, P., DANZlN, C., JUNO, i . J . , CASARA,P., VEVERT,J. P. Catalytic irreversible inhibition of mammalian ornithine decarboxylase (EC 4.1.1.17) by substrate and product analogues. J Am Chem Soc 100, 2551-2553 (1978). NAWATA,H., YAMAMOTO,R. S., POIRIER, L. A. Ornithine decarboxylase induction and polyamine levels in the kidney ofestradiol-treated castrated male rats. Life Sci 26, 689-698 (1980). REUTER,H. A. Localization offl-adrenergic receptors and effects ofnoradrenaline and cyclic nucleotides on action potentials, ionic currents and tension in mammalian cardiac muscle.J Physio1242, 429-451 (1974). REUTER,H. A. Properties of two inward membrane currents in the heart. Ann Rev Physio141,413 424 (1979). REUTER, H. A. Calcium channel modulation by neurotransmitters, enzymes and drugs. Nature 301, 569-374 (1983). RINALDI,M. L., CAPON'Z,J.-P., DEMAILLE,J. G. The cyclic AMP-dependent modulation of cardiac sarcolemmal slow calcium channels. J Mol Cell Cardio114, 279-289 (1982). STEINMAN,R. M., COHN, J. A. The interaction of soluble horseradish peroxidase with mouse peritoneal macrophages in vitro.J Cell Bio155, 18f~204 (1972). TABOR,C. W., TABOR, H. Polyamines. Ann Rev Biochem 53, 749 790 (1984). TSIEN,R. W. Cyclic AMP and contractile activity in heart. Adv Cyclic Nucleotide Res 8, 363-420 (1977). VAN BREEMEN, C., CASTEELS, R. The use of Ca-EGTA in measurements of *SCa effiux from smooth muscle. Pfliigers Arch 348, 239-245 (1974). WARNICA,J. W., A~TONY, P., GiBsoN, K., HARMS, P. The effect ofisoprenaline and propranolol on rat myocardial ornithine decarboxylase. Cardiovasc Res 9, 793-796 (1975).