Brain Research, 542 (1991) 141-146
141
Elsevier BRES 24538
Spinal cord ischemia reduces calcium/calmodulin-dependent protein kinase activity Abha Kochhar 1, Tsunao Saitoh 1'2 and Justin A. Z i v i n 1'3 t Department of Neurosciences, 0624, School of Medicine, University of California, San Diego, La Jolla, CA 92093 (U.S.A.) and 2Centerfor Molecular Genetics, University of California, San Diego, La Jolla, CA 92093-0624 (U.S.A.) and 3Neurology Service, San Diego Veterans Administration Medical Center, San Diego, CA 92161 (U.S.A.)
(Accepted 13 November 1990) Key words: Calcium/calmodulin-dependentprotein kinase; Ischemia; Phosphorylation; Spinal cord; Neurodegeneration
Calcium/calmodulin (Ca2+/CaM)-dependent protein phosphorylation was evaluated in a rabbit spinal cord ischemia model. One hour of ischemia reduced particulate (5% of control) and cytosolic (35% of control) Ca2+/CaM-dependent protein kinase activity significantly (p < 0.01). In vitro phosphorylation of endogenous proteins by endogenous Ca2+/CaM-dependent protein kinase showed that phosphorylation of 14 particulate and 7 cytosolic proteins was stimulated in the presence of Ca2÷/CaM in control tissue. However, after 1 hour of ischemia, Ca2+/CaM-dependent protein phosphorylation was virtually absent in the particulate fraction and significantly reduced in the cytosol. When equal amounts of control and ischemic tissue samples were combined and assayed, Ca2+/CaM-dependent protein kinase activity was 43% of control in particulate and 70% of control in cytosolic fractions. This suggests that reduced Ca2+/CaM-dependent protein phosphorylation is probably not due to the presence of an inhibitory activity in ischemic tissue. These results show that the Ca2÷/CaM-dependent protein phosphorylation system is impaired after ischemia durations which cause irreversible damage. These altered phosphorylation reactions may play critical roles in mediating irreversible neurologic injury,
The specific biochemical events leading to loss of integrated neurologic function after irreversible ischemia are still unknown, although, several sequences of events occurring during ischemia have been identified 11. Energy-producing processes are severely affected, resulting in rapid depletion of ATP 12. Protein synthesis is depressed 3 and intracellular calcium homeostasis is disrupted 4,9,11. Many extracellular signals regulate intracellular events by initiating a transient rise in the cytosolic calcium concentration. Calcium is an important intracellular regulator of many different cell processes and enzymes. Many of its actions appear to be mediated by calciumdependent protein phosphorylation 8. The initial ischemic events, depleted energy stores and elevated intraceUular calcium levels, could have profound effects on calciumdependent protein phosphorylation. We found that drugs which interfere with calcium-dependent protein phosphorylation (phenothiazines) reduce neurological deficits produced by focal CNS ischemia ~8. However, phenothiazines have many additional effects which may indirectly affect calcium-dependent protein phosphorylation. We have also shown that calcium/phospholipid-dependent protein phosphorylation is selectively impaired during
irreversible CNS ischemia 6. In the present study, we investigated the effects of irreversible ischemia on Ca2+/ CaM-dependent protein phosphorylation in rabbit spinal cord. The rabbit spinal cord ischemia model (RSCIM) provides a convenient method for producing highly reproducible lesions in the caudal spinal cord, without affecting more rostral regions 16. We used the RSCIM to evaluate in vitro protein phosphorylation under Ca2÷/ CaM-dependent protein kinase-activating conditions in ischemic and control spinal cord. Spinal cord ischemia was produced by occluding the aorta just caudal to the renal arteries in New Zealand White rabbits. Male rabbits weighing 2-3 kg were anesthetized with halothane and a small-diameter Tygon tube (Norton Co., Akron, OH) was placed around the aorta. The ends of the line were threaded through a small plastic button and then through a larger diameter Tygon tube to form a snare ligature. The incision was closed around the tubing so that the free ends were accessible externally. Animals were allowed to recover from anesthesia for at least 3 h. The aorta was occluded by pulling and clamping the small tubing for 1 h in fully awake rabbits. All of these rabbits became paraplegic within 2 min after the onset of
Correspondence: J. Zivin , Department of Neurosciences, 0624, School of Medicine, University of California, San Diego, La Jolla, CA 92093-0624, U.S.A.
142 occlusion. One hour of ischemia produces irreversible paralysis in essentially all rabbits 16. A snare ligature was positioned around the aorta but it was not clamped in control rabbits. At the end of the hour, animals were sacrificed with E U T H A - V I (Anthony Products Co., Arcadia, CA). The spinal column from the costovertebral junction to the sacrum was removed en bloc and the spinal cord was quickly pushed out using a metal plunger with a rubber tip. The spinal cord was rapidly frozen in dry ice, and cut in the middle of the lumbar enlargement and between the upper lumbar and thoracic regions. The low lumbar/ sacral segment and the thoracic segment were collected and stored at -70 °C. In occluded rabbits, the low lumbar/sacral cord is ischemic and the thoracic cord is non-ischemic. The low lumbar/sacral segment from nonoccluded rabbits is referred to as control tissue. Tissue samples were homogenized in 10 volumes of homogenization buffer (0.32 M sucrose; 5 mM HEPES, pH 8; 5 mM benzamidine; 2 mM fl-mercaptoethanol; 3 mM EGTA; 0.5 mM MgSO4; 0.1 mM phenylmethylsulfonyl fluoride (pMSF); 0.1 mg/ml leupeptin; 0.05 mg/ml pepstatin; and 0.1 mg/ml aprotinin). Homogenates were centrifuged for 1 h at 100,000 g at 2 °C to separate the particulate fraction (100,000 g pellet) from the cytosol. The pellet was reconstituted in the original volume of homogenization buffer. Protein concentrations in the cytosolic and particulate fractions were determined using the method of Lowry et al. 7 with plasma globulin (Bio-Rad, Richmond, CA) as the standard protein. Each fraction (containing 6.3 /~g total protein) was incubated in a reaction mixture containing 50 mM Tris-HCl, pH 7.6; 10 mM MgSO4; 0.2 mM EDTA; 5 mM fl-mercaptoethanol; 10 ~tM ATP; 1/~Ci [3~p]ATP (ICN, lrvine, CA); and 100 ~tg/ml PK-A inhibitor, in the presence or absence of specific activators. Ca2+/CaM dependent protein phosphorylation was assessed in the presence of 1.5 mM CaCI2 and 20 /~g/mi calmodulin. Protein kinase activity was determined by adding 100/~M of Ca2+/CaM protein kinase-specific substrate 1°, a synthetic decapeptide, Pro-Leu-Arg-Arg-Thr-Leu-Ser-ValAIa-Ala-NH 2 (UCSD, La Jolla, CA), to the reaction mixture in the presence or absence of activators. Reactions were incubated 1 min at 30 °C. For endogenous protein phosphorylation, the reaction was stopped by addition of 6.3/~1 of buffer containing 40% (v/v) glycerol, 25% fl-mercaptoethanol, 12% SDS, 0.31 M Tris-HCI (pH 6.8), 25 mM EDTA, and 0.1% Bromophenol blue. For Ca2+/CaM-dependent protein kinase activity assays, the reaction was stopped by spotting the reaction on phosphoceUulose, P81 paper (Whatman, Hillsboro, OR), and immediately washing the filters in 75 mM phosphoric acid. Calmodulin and PK-A inhibitor are from Sigma
FABLE I Calcium/calrnodulin-dependent protein kinase activity in lumbar spinal cord
Kinase activity was assayed under standard assay conditions as described in the text except that 100 /~M peptide substrate (Pro-Leu-Arg-Arg-Thr-Leu-Ser-Val-Ala-Ala-NHz)was included in the reaction. Activator-dependent incorporation of 32p into the peptide substrate was quantified by phosphocellulose filter assay and liquid scintillation counting. Equal aliquots of control and ischemic tissue were combined to determine kinase activity of the mixture (control + ischemic). Values represent means + S.E.M. (n = 4). Peptide substrate phosphorylation (pmol/mg peptide substrate/min)
Control Ischemic Control + ischemic
Particulate
Cytosol
528 + 62 27 + 22** 225 + 2**
417 + 89 146 + 35** 291 + 39
** Significantly different from control (P < 0.01).
Chemical Co. (St. Louis, MO). Phosphoproteins, labeled with [3zp]ATE were separated by gradient (6.5-16%) SDS-polyacrylamide gel electrophoresis. Gels were fixed in 10% acetic acid/15% isopropyl alcohol, stained with 0.2% Coomassie brilliant blue in 10% acetic acid/40% isopropyl alcohol, and destained in 10% acetic acid/15% isopropyl alcohol. Destained gels were rinsed with water, dried, and exposed to X-Omat RP film (Eastman Kodak Co., Rochester, NY) with Hi Plus intensifying screen (Du Pont Co., Wilmington, DE) at -70 °C. The relative intensity of each band was quantified using densitometric analysis of the autoradiograms. Peak areas for individual phosphoprotein bands were used to calculate phosphorylation ratios for endogenous substrates in the in vitro phosphorylation assays. Substrate phosphorylation ratios express the relative amounts of phosphorylation in the presence of activators divided by the amount of phosphorylation in the absence of activators. Basal phosphorylation is the sum of all phosphoprotein band peak areas in the absence of activators, and total phosphorylation is the sum of peak areas in the presence of activators. Data were analyzed by analysis of variance with repeated measures and individual comparisons were made using the Newman-Keuls method TM- Comparisons between the arithmetic average of the individual phosphorylation ratios of control and ischemic tissue and the ratio of the combined tissue samples were made using the sign test TM. Ca2+/CaM-dependent protein kinase activity was assayed in ischemic and control tissue using a synthetic decapeptide as the substrate (Table I). Kinase activity was expressed as the amount of 32p incorporated per mg protein in tissue homogenates per min. Ca2÷/CaM -
143 tic scans. Addition of Ca2+/CaM enhanced total endogenous protein phosphorylation (sum of all phosphoprotein peak areas) only 52% in ischemic tissue (3.53 + 0.21), and 307% in control tissue (17.5 + 1.28). The stimulation by Ca2+/CaM was significantly reduced in ischemic tissue (P < 0.01). In the control cytosolic fraction, addition of CaE+/CaM stimulated phosphorylation of M r 22,000 and 31,000 proteins to the greatest extent, followed by M r 24,000, 46,000, 57,000 and 80,000 protein phosphorylation (Fig. 1 and Table II). Phosphorylation of the M r 34,000 protein was slightly stimulated in the presence of Ca2+/CaM in control cytosol. In the cytosolic fraction, the effect of ischemia on endogenous protein phosphorylation was not as pronounced as in the particulate fraction. After 1 h of ischemia, endogenous phosphorylation of 5 of 7 cytosolic proteins was reduced significantly in the presence of CaE+/CaM (Fig. 1 and Table II). Phosphorylation of M r 34,000 and 57,000 proteins was almost absent in ischemic cytosolic samples, while M r 22,000, 31,000 and 80,000 protein phosphorylation was reduced to approximately 60% of control. M r 24,000 and 46,000 protein phosphorylation was not affected by ischemia. In contrast to particulate samples, basal phosphorylation was not re-
dependent protein kinase activity in ischemic tissue was 3% of control tissue in lumbar particulate fractions and 35% of controls in the cytosol. In vitro phosphorylation of several endogenous protein bands was stimulated in the presence of Ca2+/CaM in the particulate fraction of control lumbar tissue (Fig. 1 and Table II). M r 57,000 protein phosphorylation showed the greatest magnitude of stimulation in the presence of Ca2+/CaM, followed by M r 24,000, 46,000, 55,000, 60,000 and 80,000 protein phosphorylation (Table II). Addition of Ca2+/CaM stimulated phosphorylation of several other proteins, M r 12,000, 17,000, 22,000, 31,000, 52,000, 120,000, 140,000 and 300,000, to a lesser extent. After 1 h of ischemia, addition of CaE+/CaM failed to enhance endogenous phosphorylation of proteins in lumbar particulate fractions, except for a slight increase in M r 22,000 and 46,000 protein phosphorylation (Table II). A similar response was observed when total protein phosphorylation was examined. Basal phosphorylation (sum of all phosphoprotein peak areas in the absence of Ca2+/CaM) was reduced significantly (P < 0.01) in ischemic particulate samples (control, 4.3 + 0.16; ischemic, 2.33 _+ 0.36; n = 4, mean + S.E.M.). These numbers represent arbitrary area units from densitomet-
CBB
C
]~
!
~','.t
CBB
II
I
C
,
C÷I
,
Mr x 10-3 200
116 92
66
45
~iiiii~il¸i~ 31""~ 31
! iiiii~iiii;
i;ii!i~;~iiiii'~iiii~,i~iill
14
14 iiiiilJl;ili!iii!iiiiii!!ili ~; C I
][
Ca+2/CaM
-
÷
-
÷
-
+
C I
]
Ca+2/CaM
-
,!, -
+
-
÷
I
CYTOSOL PARTICULATE Fig. 1. Reduced calcium/calmodulin-dependentprotein phosphorylationin ischemiclumbar spinal cord. Coomassie brilliant blue (CBB) staining of gels with 6.3/zg protein per lane is shown for control (C) and ischemic(1) lumbar spinal cord rabbits. Typicalautoradiograms of 32p-labeled proteins from cytosolic and particulate fractions in the absence of activators, calcium/calmodulin (- lane), or in the presence of calcium/calmodulin (+ lane) are shown for control (C), ischemic (I), and mixture of control and ischemic samples (C+I). The arrows show Ca2+/CaM-dependent phosphoproteins in control tissue.
144 TABLE
II
In vitrophosphorylation of endogenous substrates by endogenous Cae+/CaM-dependent protein kinase in lumbar spinal cord Substrate phosphorylation ratios express the relative amounts of phosphorylation in the presence of activators (calcium/calmodulin) divided by the phosphorylation in the absence of activators. Values represent means _+ S.E.M. (n = 4).
k Da
Phosphorylation Particulate
300 140 120 80 60 57 55 52 46 34 31 24 22 17 12 Total b
Cytosol
Control
lschemic
Combined
Control
lschemic
Combined
2.26 + 0.84 3.42+1.32 3.54 + 0.92 5.13+0.51 5.22 + 0.57 20.48 + 0.97 5.87 + 0.44 3.84 _+0.22 5.64 + 0.71 2.53 + 0.99 7.16 _+0.43 2.47 _+0.23 2.10+0.30 3.66 _+0.27
0.50 _+0.08** 0.75_+0.11"* 0.79 _+0.12"* 1.25+0.13"* 0.54 + 0.22** 0.69 _+0.23** 0.76 _+0.09** 0.61 + 0.18"* 1.87 + 0.34** 0.65 + 0.35** 1.29 + 0.44** 1.79 + 0.26** 0.10+0.09"* 0.56 + 0.31"*
1.28 + 0.28 1.79+0.12 2.21 _+ 0.40 3.38+0.28 2.94 + 1.03 11.04 + 0.54 2.59 + 0.39 2.60 + 0.54 3.50 _+0.11 0.47 + 0.28*** 2.74 + 0.07*** 3.22 + 0.05 2.46+0.21 2.33 + 1.18
_a 8.75+0.90 5.30 + 0.83 7.37 + 0.98 4.36 + 0.80 29.35 _+2.93 9.03 + 0.70 18.85 + 3.61 -
_ 5.21+0.11" 1.87 + 0.35** 5.05 + 0.80 1.68 + 0.21"* 18.32 + 2.94** 9.56 + 1.09 11.82 + 0.43* -
_ 8.47+0.87 4.20 _+0.25 7.55 + 0.56 2.11 + 0.24 25.68 + 1.88 9.18 + 0.79 17.63 + 1.39
4.63 + 0.27
1.59 + 0.20**
10.70 _+ 1.00
5.82 + 0.70**
9.39 + 0.24
3.00 + 0.12
a Not detectable. b Total protein phosphorylation ratio express the amount of phosphorylation in the total lane in the presence of calcium/calmodulin divided by phosphorylation in the total lane in the absence of calcium/calmodulin. * Significantly different from control (P < 0.05). ** Significantly different from control (P < 0.01). * * * Significantly less than the arithmetic average of control and ischemic value.
duced
in
ischemic
cytosolic
samples
(1.55
+
0.18;
W h e n e q u a l aliquots of c o n t r o l and i s c h e m i c l u m b a r
c o n t r o l s , 1.40 + 0.08). In t h e p r e s e n c e of C a 2 + / C a M ,
tissue
total p r o t e i n p h o s p h o r y l a t i o n in i s c h e m i c cytosolic sam-
kinase
ples (6.14 ___ 0.44) was significantly l o w e r than control
particulate
cytosol (14.98 + 1.10, P < 0.01). It is not likely that the r e d u c e d C a Z + / C a M - d e p e n d e n t
cytosolic f r a c t i o n (Table I). A similar r e s p o n s e was f o u n d
p r o t e i n kinase p h o s p h o r y l a t i o n in i s c h e m i c tissue is due
tissue (Fig.
to a l t e r e d substrate
ischemic particulate
availability, since p r o t e i n
kinase
were
combined,
activity i n c r e a s e d fraction
and
Ca2+/CaM-dependent
protein
to 4 3 %
o f c o n t r o l s in the
to 7 0 %
of c o n t r o l s
in the
in substrate p h o s p h o r y l a t i o n ratios in c o m b i n e d l u m b a r 1 and T a b l e II). C o m b i n e d samples
yielded
c o n t r o l and
substrate
phos-
activity is r e d u c e d e v e n w h e n an e x o g e n o u s substrate (10
p h o r y l a t i o n ratios that w e r e close to o r g r e a t e r t h a n the
a m i n o acid p e p t i d e ) is a d d e d to tissue samples. O t h e r possible e x p l a n a t i o n s for i m p a i r e d C a 2 + / C a M - d e p e n d e n t
a r i t h m e t i c a v e r a g e of individual s a m p l e s , e x c e p t for M r 24,000 and 31,000 p r o t e i n p h o s p h o r y l a t i o n ratios, which
p h o s p h o r y l a t i o n are r e d u c e d p r o t e i n kinase levels d u e to
w e r e closer to i s c h e m i c v a l u e s t h a n to the a r i t h m e t i c
enzyme
degradation
or
generation
of
an
inhibitory
a v e r a g e of t h e t w o s a m p l e s (Table II). All substrate
activity in i s c h e m i c tissue 13. If an inhibitory activity is
phosphorylation
p r e s e n t in i s c h e m i c tissue, the m a g n i t u d e of p h o s p h o r y -
close to c o n t r o l values. Similar results w e r e o b t a i n e d in
lation in a m i x t u r e o f e q u a l a m o u n t s of control and
total p r o t e i n p h o s p h o r y l a t i o n in p a r t i c u l a t e and cytosolic fractions. Total p r o t e i n p h o s p h o r y l a t i o n in m i x e d c o n t r o l
ischemic tissue w o u l d be less t h a n the a v e r a g e o f e a c h individual reaction. If no inhibitors w e r e p r e s e n t , and
ratios in the c o m b i n e d
cytosol w e r e
and i s c h e m i c samples was 6 5 % o f c o n t r o l (11.34 + 1.54)
a s s u m i n g no i n t e r a c t i o n s , we w o u l d e x p e c t C a 2 + / C a M -
in the p a r t i c u l a t e fraction and 8 7 % of c o n t r o l (13.00 +
d e p e n d e n t kinase activity or substrate p h o s p h o r y l a t i o n ratios (relative a m o u n t s of p h o s p h o r y l a t i o n in the prese n c e of C a 2 + / C a M d i v i d e d by p h o s p h o r y l a t i o n in the
0.50) in the cytosol. This s u g g e s t e d that no inhibitory
a b s e n c e of activators) of the m i x t u r e to b e the a r i t h m e t i c a v e r a g e of individual control and ischemic samples.
activity was p r e s e n t in i s c h e m i c tissue. T h e in v i t r o p h o s p h o r y l a t i o n p a t t e r n of e n d o g e n o u s p r o t e i n s by e n d o g e n o u s C a 2 + / C a M - d e p e n d e n t p r o t e i n kinase in the thoracic r e g i o n of the spinal c o r d was
145 TABLE III In vitro phosphorylation of endogenous substrates by endogenous Ca3+/CaM.dependent protein kinase in thoracicspinal cord
Substrate phosphorylation ratios express the relative amounts of phosphorylation in the presence of activators (calcium/calmodulin) divided by the phosphorylation in the absence of activators. Values represent means + S.E.M. (n = 4). kDa
Substrate phosphorylation ratios Particulate
Cytosol
Control
Ischemic
Control
lschemic
300 120 80 60 57 55 46 24 22 12
2.48 + 0.56 3.26 + 0.41 2.56 + 0.44 4.11 _ 0.68 8.61 + 0.05 3.51 + 1.20 2.88 + 0.04 8.89 _+ 1.00 1.91 + 0.42 4.68 + 0.53
3.84 + 0.01 3.68 + 0.95 2.25 + 0.08 4.65 + 0.55 7.50 + 0.21 2.88 + 0.06 3.85+ 0.31 8.49 ___0.12 2.23 + 0.67 3.84 + 0.01
_a 4.16 + 0.66 6.69 + 0.67 2.47 + 0.82 -
_ 3.33+ 0.16 8.65+ 0.34 1.91 +_0.25 -
Totalb
2.77 + 0.06
2.62 + 0.38
3.63+ 0.44
2.20 + 0.26
a Not detectable. b Total protein phosphorylation ratio expresses the amount of phosphorylation in the total lane in the presence of calcium/ calmodulin divided by phosphorylation in the total lane in the absence of calcium/calmodulin.
+ 2.59 in control particulate samples, and 23.02 + 2.02 in samples from ischemic animals. In thoracic cytosol, basal phosphorylation was 7.21 + 1.57 in controls, and 7.51 + 0.46 in ischemic animals; and activated phosphorylation was 17.26 + 0.79 in controls, and 16.37 + 0.88 in ischemic animals. These results show that altered Ca2+/CaM-depen dent protein kinase phosphorylation reactions are limited to the ischemic region of the spinal cord. In the present study, we found that the Ca2+/CaMdependent protein kinase phosphorylation system was impaired during irreversible ischemia in the rabbit spinal cord. Two other studies have examined the effects of ischemia on Ca2+/CaM-mediated protein phosphorylation during ischemia and recovery in the gerbil hippocampus 1'13. Both studies show that in vitro phosphorylation of endogenous substrates by endogenous Ca2+/CaM-dependent protein kinase was reduced when circulation was restored for 2 h after a 5 rain ischemic insult in adult gerbil hippocampus. This reduction in Ca2÷/CaM-dependent protein phosphorylation persisted after recirculation for 7 days. This implies that Cae+/CaM-dependent protein kinase activity may be susceptible to permanent inactivation in vivo after brief episodes of cerebral ischemia. However, it is still uncertain whether this inactivation is an initiating event in the pathogenesis of ischemic cell damage or an end result of irreversible ischemic injury. To intervene in the neuronal death caused by ischemia, it is important to understand the molecular mechanisms which render neurons unable to recover from ischemic stress. To study this process of neuronal death, it is essential to identify the biochemical reactions which are involved in the physiological changes during the critical period. Decreased Ca2÷/CaM-dependent protein kinase activity may be an important marker to follow the ischemic process. We have previously shown that the c A M P - d e p e n d e n t protein phosphorylation reactions seem to be intact in the critical phases of ischemic injury, while calcium/phospholipid-dependent protein phosphorylation is selectively impaired after irreversible CNS ischemia in the rabbit spinal cord 17. These findings suggest that aberrant calcium-dependent phosphorylation reactions are specific changes of the ischemic process, and not the result of derangement of general protein phosphorylation.
evaluated in control and ischemic rabbits to verify that r e d u c e d C a 2 + / C a M - d e p e n d e n t protein phosphorylation was limited to ischemic regions of the spinal cord. A o r t i c occlusion in ischemic rabbits does not affect thoracic spinal cord perfusion (non-ischemic). The Ca2÷/CaM d e p e n d e n t p r o t e i n p h o s p h o r y l a t i o n p a t t e r n in thoracic particulate tissue was similar to l u m b a r tissue, except the magnitude of p h o s p h o r y l a t i o n was lower in thoracic tissue (Table III). M i n o r p h o s p h o p r o t e i n s (M r 17,000, 31,000, 52,000 and 140,000) were absent in the particulate fraction from thoracic spinal cord. Cytosolic protein p h o s p h o r y l a t i o n profiles were different in thoracic and l u m b a r tissue. A d d i t i o n of Ca2+/CaM only stimulated p h o s p h o r y l a t i o n of 3 cytosolic proteins, M r 80,000, 44,000 and 21,000, in thoracic tissue. In vitro phosphorylation of e n d o g e n o u s proteins by endogenous Ca2+/ C a M - d e p e n d e n t protein kinase was similar in control and ischemic thoracic spinal cord. Basal phosphorylation was 7.48 + 1.10, n = 4, in control particulate samples, and 8.96 + 1.29, n = 4, in ischemic particulate samples. Total p h o s p h o r y l a t i o n in the presence of Ca2+/CaM was 20.66
This research was supported by research grants from the National Institutes of Health (NS 23814 and NS 28121) and the McKnight Endowment Fund for Neuroscience.
1 Bodsch, W., Takahashi, A., Barbier, A., Grosse Ophoff, B. and Hossmann, K.-A., Cerebral protein synthesis and ischemia, Prog. Brain Res., 63 (1985) 197-210. 2 Omitted. 3 Cooper, H.K., Zalewska, T., Kawakami, S., Hossmann, K.-A. and Kleihues, P., The effect of ischaemia and recirculation on
protein synthesis in the rat brain, J. Neurochem., 28 (1977) 929-934. 4 Harris, R.J., Symon, L., Branston, N.M. and Bayhan, M., Changes in extracellular calcium activity in cerebral ischaemia, J. Cereb. Blood Flow. Metab., 1 (1981) 203-209. 5 Omitted.
146 6 Kochhar, A., Saitoh, T. and Zivin, J., Reduced protein kinase C activity in ischemic spinal cord, J. Neurochem., 53 (1989) 946--953. 7 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-272. 8 Nairn, A.C., Hemmings, H.C., Jr. and Greengard, P., Protein kinases in the brain, Annu. Rev. Biochem., 54 (1985) 931-976. 9 Nowicki, J.P., MacKenzie, E.T. and Young, A.R., Brain ischaemia, calcium and calcium antagonists, Pathol. Biol., 30 (1982) 282-288. 10 Pearson, R.B., Woodgett, J.R., Cohen, P. and Kemp, B.E., Substrate specificity of a multifunctional calmodulin-dependent protein kinase, J. Biol. Chem., 260 (1985) 14471-14476. 11 Raichle, M.E., The pathophysiology of brain ischemia, Ann. Neurol., 13 (1983) 2-10. 12 Siesj6, B.K. and Ljunggren, B., Cerebral energy reserve after prolonged hypoxia and ischemia, Arch. Neurol., 29 (1973)
40O-4O3. 13 Taft, W.C., Tennes-Rees, K.A., Blair, R.E., Clifton, G.L. and DeLorenzo, R.J., Cerebral ischemia decreases endogenous calcium-dependent protein phosphorylation in gerbil brain, Brain Research, 447 (1988) 159-163. 14 Winer, B.J., Statistical Principles in Experimental Design, 2nd edn., McGraw-Hill, New York, 1971. 15 Omitted. 16 Yatsu, F.M., Lee, L.-W. and Liao, C.-L., Energy metabolism during brain ischemia. Stability during reversible and irreversible damage, Stroke, 6 (1975) 678-683. 17 Zivin, J.A., DeGirolami, U. and Hurwitz, E.L., A quantitative study of the spectrum of neurological deficits in experimental CNS ischemia, Arch. Neurol., 39 (1982) 408-412. 18 Zivin, J.A., Kochhar, A. and Saitoh, T., Phenothiazines reduce ischemic damage to the central nervous system, Brain Research, 48 (1989) 189-193.
141
Elsevier BRES 24538
Spinal cord ischemia reduces calcium/calmodulin-dependent protein kinase activity Abha Kochhar 1, Tsunao Saitoh 1'2 and Justin A. Z i v i n 1'3 t Department of Neurosciences, 0624, School of Medicine, University of California, San Diego, La Jolla, CA 92093 (U.S.A.) and 2Centerfor Molecular Genetics, University of California, San Diego, La Jolla, CA 92093-0624 (U.S.A.) and 3Neurology Service, San Diego Veterans Administration Medical Center, San Diego, CA 92161 (U.S.A.)
(Accepted 13 November 1990) Key words: Calcium/calmodulin-dependentprotein kinase; Ischemia; Phosphorylation; Spinal cord; Neurodegeneration
Calcium/calmodulin (Ca2+/CaM)-dependent protein phosphorylation was evaluated in a rabbit spinal cord ischemia model. One hour of ischemia reduced particulate (5% of control) and cytosolic (35% of control) Ca2+/CaM-dependent protein kinase activity significantly (p < 0.01). In vitro phosphorylation of endogenous proteins by endogenous Ca2+/CaM-dependent protein kinase showed that phosphorylation of 14 particulate and 7 cytosolic proteins was stimulated in the presence of Ca2÷/CaM in control tissue. However, after 1 hour of ischemia, Ca2+/CaM-dependent protein phosphorylation was virtually absent in the particulate fraction and significantly reduced in the cytosol. When equal amounts of control and ischemic tissue samples were combined and assayed, Ca2+/CaM-dependent protein kinase activity was 43% of control in particulate and 70% of control in cytosolic fractions. This suggests that reduced Ca2+/CaM-dependent protein phosphorylation is probably not due to the presence of an inhibitory activity in ischemic tissue. These results show that the Ca2÷/CaM-dependent protein phosphorylation system is impaired after ischemia durations which cause irreversible damage. These altered phosphorylation reactions may play critical roles in mediating irreversible neurologic injury,
The specific biochemical events leading to loss of integrated neurologic function after irreversible ischemia are still unknown, although, several sequences of events occurring during ischemia have been identified 11. Energy-producing processes are severely affected, resulting in rapid depletion of ATP 12. Protein synthesis is depressed 3 and intracellular calcium homeostasis is disrupted 4,9,11. Many extracellular signals regulate intracellular events by initiating a transient rise in the cytosolic calcium concentration. Calcium is an important intracellular regulator of many different cell processes and enzymes. Many of its actions appear to be mediated by calciumdependent protein phosphorylation 8. The initial ischemic events, depleted energy stores and elevated intraceUular calcium levels, could have profound effects on calciumdependent protein phosphorylation. We found that drugs which interfere with calcium-dependent protein phosphorylation (phenothiazines) reduce neurological deficits produced by focal CNS ischemia ~8. However, phenothiazines have many additional effects which may indirectly affect calcium-dependent protein phosphorylation. We have also shown that calcium/phospholipid-dependent protein phosphorylation is selectively impaired during
irreversible CNS ischemia 6. In the present study, we investigated the effects of irreversible ischemia on Ca2+/ CaM-dependent protein phosphorylation in rabbit spinal cord. The rabbit spinal cord ischemia model (RSCIM) provides a convenient method for producing highly reproducible lesions in the caudal spinal cord, without affecting more rostral regions 16. We used the RSCIM to evaluate in vitro protein phosphorylation under Ca2÷/ CaM-dependent protein kinase-activating conditions in ischemic and control spinal cord. Spinal cord ischemia was produced by occluding the aorta just caudal to the renal arteries in New Zealand White rabbits. Male rabbits weighing 2-3 kg were anesthetized with halothane and a small-diameter Tygon tube (Norton Co., Akron, OH) was placed around the aorta. The ends of the line were threaded through a small plastic button and then through a larger diameter Tygon tube to form a snare ligature. The incision was closed around the tubing so that the free ends were accessible externally. Animals were allowed to recover from anesthesia for at least 3 h. The aorta was occluded by pulling and clamping the small tubing for 1 h in fully awake rabbits. All of these rabbits became paraplegic within 2 min after the onset of
Correspondence: J. Zivin , Department of Neurosciences, 0624, School of Medicine, University of California, San Diego, La Jolla, CA 92093-0624, U.S.A.
142 occlusion. One hour of ischemia produces irreversible paralysis in essentially all rabbits 16. A snare ligature was positioned around the aorta but it was not clamped in control rabbits. At the end of the hour, animals were sacrificed with E U T H A - V I (Anthony Products Co., Arcadia, CA). The spinal column from the costovertebral junction to the sacrum was removed en bloc and the spinal cord was quickly pushed out using a metal plunger with a rubber tip. The spinal cord was rapidly frozen in dry ice, and cut in the middle of the lumbar enlargement and between the upper lumbar and thoracic regions. The low lumbar/ sacral segment and the thoracic segment were collected and stored at -70 °C. In occluded rabbits, the low lumbar/sacral cord is ischemic and the thoracic cord is non-ischemic. The low lumbar/sacral segment from nonoccluded rabbits is referred to as control tissue. Tissue samples were homogenized in 10 volumes of homogenization buffer (0.32 M sucrose; 5 mM HEPES, pH 8; 5 mM benzamidine; 2 mM fl-mercaptoethanol; 3 mM EGTA; 0.5 mM MgSO4; 0.1 mM phenylmethylsulfonyl fluoride (pMSF); 0.1 mg/ml leupeptin; 0.05 mg/ml pepstatin; and 0.1 mg/ml aprotinin). Homogenates were centrifuged for 1 h at 100,000 g at 2 °C to separate the particulate fraction (100,000 g pellet) from the cytosol. The pellet was reconstituted in the original volume of homogenization buffer. Protein concentrations in the cytosolic and particulate fractions were determined using the method of Lowry et al. 7 with plasma globulin (Bio-Rad, Richmond, CA) as the standard protein. Each fraction (containing 6.3 /~g total protein) was incubated in a reaction mixture containing 50 mM Tris-HCl, pH 7.6; 10 mM MgSO4; 0.2 mM EDTA; 5 mM fl-mercaptoethanol; 10 ~tM ATP; 1/~Ci [3~p]ATP (ICN, lrvine, CA); and 100 ~tg/ml PK-A inhibitor, in the presence or absence of specific activators. Ca2+/CaM dependent protein phosphorylation was assessed in the presence of 1.5 mM CaCI2 and 20 /~g/mi calmodulin. Protein kinase activity was determined by adding 100/~M of Ca2+/CaM protein kinase-specific substrate 1°, a synthetic decapeptide, Pro-Leu-Arg-Arg-Thr-Leu-Ser-ValAIa-Ala-NH 2 (UCSD, La Jolla, CA), to the reaction mixture in the presence or absence of activators. Reactions were incubated 1 min at 30 °C. For endogenous protein phosphorylation, the reaction was stopped by addition of 6.3/~1 of buffer containing 40% (v/v) glycerol, 25% fl-mercaptoethanol, 12% SDS, 0.31 M Tris-HCI (pH 6.8), 25 mM EDTA, and 0.1% Bromophenol blue. For Ca2+/CaM-dependent protein kinase activity assays, the reaction was stopped by spotting the reaction on phosphoceUulose, P81 paper (Whatman, Hillsboro, OR), and immediately washing the filters in 75 mM phosphoric acid. Calmodulin and PK-A inhibitor are from Sigma
FABLE I Calcium/calrnodulin-dependent protein kinase activity in lumbar spinal cord
Kinase activity was assayed under standard assay conditions as described in the text except that 100 /~M peptide substrate (Pro-Leu-Arg-Arg-Thr-Leu-Ser-Val-Ala-Ala-NHz)was included in the reaction. Activator-dependent incorporation of 32p into the peptide substrate was quantified by phosphocellulose filter assay and liquid scintillation counting. Equal aliquots of control and ischemic tissue were combined to determine kinase activity of the mixture (control + ischemic). Values represent means + S.E.M. (n = 4). Peptide substrate phosphorylation (pmol/mg peptide substrate/min)
Control Ischemic Control + ischemic
Particulate
Cytosol
528 + 62 27 + 22** 225 + 2**
417 + 89 146 + 35** 291 + 39
** Significantly different from control (P < 0.01).
Chemical Co. (St. Louis, MO). Phosphoproteins, labeled with [3zp]ATE were separated by gradient (6.5-16%) SDS-polyacrylamide gel electrophoresis. Gels were fixed in 10% acetic acid/15% isopropyl alcohol, stained with 0.2% Coomassie brilliant blue in 10% acetic acid/40% isopropyl alcohol, and destained in 10% acetic acid/15% isopropyl alcohol. Destained gels were rinsed with water, dried, and exposed to X-Omat RP film (Eastman Kodak Co., Rochester, NY) with Hi Plus intensifying screen (Du Pont Co., Wilmington, DE) at -70 °C. The relative intensity of each band was quantified using densitometric analysis of the autoradiograms. Peak areas for individual phosphoprotein bands were used to calculate phosphorylation ratios for endogenous substrates in the in vitro phosphorylation assays. Substrate phosphorylation ratios express the relative amounts of phosphorylation in the presence of activators divided by the amount of phosphorylation in the absence of activators. Basal phosphorylation is the sum of all phosphoprotein band peak areas in the absence of activators, and total phosphorylation is the sum of peak areas in the presence of activators. Data were analyzed by analysis of variance with repeated measures and individual comparisons were made using the Newman-Keuls method TM- Comparisons between the arithmetic average of the individual phosphorylation ratios of control and ischemic tissue and the ratio of the combined tissue samples were made using the sign test TM. Ca2+/CaM-dependent protein kinase activity was assayed in ischemic and control tissue using a synthetic decapeptide as the substrate (Table I). Kinase activity was expressed as the amount of 32p incorporated per mg protein in tissue homogenates per min. Ca2÷/CaM -
143 tic scans. Addition of Ca2+/CaM enhanced total endogenous protein phosphorylation (sum of all phosphoprotein peak areas) only 52% in ischemic tissue (3.53 + 0.21), and 307% in control tissue (17.5 + 1.28). The stimulation by Ca2+/CaM was significantly reduced in ischemic tissue (P < 0.01). In the control cytosolic fraction, addition of CaE+/CaM stimulated phosphorylation of M r 22,000 and 31,000 proteins to the greatest extent, followed by M r 24,000, 46,000, 57,000 and 80,000 protein phosphorylation (Fig. 1 and Table II). Phosphorylation of the M r 34,000 protein was slightly stimulated in the presence of Ca2+/CaM in control cytosol. In the cytosolic fraction, the effect of ischemia on endogenous protein phosphorylation was not as pronounced as in the particulate fraction. After 1 h of ischemia, endogenous phosphorylation of 5 of 7 cytosolic proteins was reduced significantly in the presence of CaE+/CaM (Fig. 1 and Table II). Phosphorylation of M r 34,000 and 57,000 proteins was almost absent in ischemic cytosolic samples, while M r 22,000, 31,000 and 80,000 protein phosphorylation was reduced to approximately 60% of control. M r 24,000 and 46,000 protein phosphorylation was not affected by ischemia. In contrast to particulate samples, basal phosphorylation was not re-
dependent protein kinase activity in ischemic tissue was 3% of control tissue in lumbar particulate fractions and 35% of controls in the cytosol. In vitro phosphorylation of several endogenous protein bands was stimulated in the presence of Ca2+/CaM in the particulate fraction of control lumbar tissue (Fig. 1 and Table II). M r 57,000 protein phosphorylation showed the greatest magnitude of stimulation in the presence of Ca2+/CaM, followed by M r 24,000, 46,000, 55,000, 60,000 and 80,000 protein phosphorylation (Table II). Addition of Ca2+/CaM stimulated phosphorylation of several other proteins, M r 12,000, 17,000, 22,000, 31,000, 52,000, 120,000, 140,000 and 300,000, to a lesser extent. After 1 h of ischemia, addition of CaE+/CaM failed to enhance endogenous phosphorylation of proteins in lumbar particulate fractions, except for a slight increase in M r 22,000 and 46,000 protein phosphorylation (Table II). A similar response was observed when total protein phosphorylation was examined. Basal phosphorylation (sum of all phosphoprotein peak areas in the absence of Ca2+/CaM) was reduced significantly (P < 0.01) in ischemic particulate samples (control, 4.3 + 0.16; ischemic, 2.33 _+ 0.36; n = 4, mean + S.E.M.). These numbers represent arbitrary area units from densitomet-
CBB
C
]~
!
~','.t
CBB
II
I
C
,
C÷I
,
Mr x 10-3 200
116 92
66
45
~iiiii~il¸i~ 31""~ 31
! iiiii~iiii;
i;ii!i~;~iiiii'~iiii~,i~iill
14
14 iiiiilJl;ili!iii!iiiiii!!ili ~; C I
][
Ca+2/CaM
-
÷
-
÷
-
+
C I
]
Ca+2/CaM
-
,!, -
+
-
÷
I
CYTOSOL PARTICULATE Fig. 1. Reduced calcium/calmodulin-dependentprotein phosphorylationin ischemiclumbar spinal cord. Coomassie brilliant blue (CBB) staining of gels with 6.3/zg protein per lane is shown for control (C) and ischemic(1) lumbar spinal cord rabbits. Typicalautoradiograms of 32p-labeled proteins from cytosolic and particulate fractions in the absence of activators, calcium/calmodulin (- lane), or in the presence of calcium/calmodulin (+ lane) are shown for control (C), ischemic (I), and mixture of control and ischemic samples (C+I). The arrows show Ca2+/CaM-dependent phosphoproteins in control tissue.
144 TABLE
II
In vitrophosphorylation of endogenous substrates by endogenous Cae+/CaM-dependent protein kinase in lumbar spinal cord Substrate phosphorylation ratios express the relative amounts of phosphorylation in the presence of activators (calcium/calmodulin) divided by the phosphorylation in the absence of activators. Values represent means _+ S.E.M. (n = 4).
k Da
Phosphorylation Particulate
300 140 120 80 60 57 55 52 46 34 31 24 22 17 12 Total b
Cytosol
Control
lschemic
Combined
Control
lschemic
Combined
2.26 + 0.84 3.42+1.32 3.54 + 0.92 5.13+0.51 5.22 + 0.57 20.48 + 0.97 5.87 + 0.44 3.84 _+0.22 5.64 + 0.71 2.53 + 0.99 7.16 _+0.43 2.47 _+0.23 2.10+0.30 3.66 _+0.27
0.50 _+0.08** 0.75_+0.11"* 0.79 _+0.12"* 1.25+0.13"* 0.54 + 0.22** 0.69 _+0.23** 0.76 _+0.09** 0.61 + 0.18"* 1.87 + 0.34** 0.65 + 0.35** 1.29 + 0.44** 1.79 + 0.26** 0.10+0.09"* 0.56 + 0.31"*
1.28 + 0.28 1.79+0.12 2.21 _+ 0.40 3.38+0.28 2.94 + 1.03 11.04 + 0.54 2.59 + 0.39 2.60 + 0.54 3.50 _+0.11 0.47 + 0.28*** 2.74 + 0.07*** 3.22 + 0.05 2.46+0.21 2.33 + 1.18
_a 8.75+0.90 5.30 + 0.83 7.37 + 0.98 4.36 + 0.80 29.35 _+2.93 9.03 + 0.70 18.85 + 3.61 -
_ 5.21+0.11" 1.87 + 0.35** 5.05 + 0.80 1.68 + 0.21"* 18.32 + 2.94** 9.56 + 1.09 11.82 + 0.43* -
_ 8.47+0.87 4.20 _+0.25 7.55 + 0.56 2.11 + 0.24 25.68 + 1.88 9.18 + 0.79 17.63 + 1.39
4.63 + 0.27
1.59 + 0.20**
10.70 _+ 1.00
5.82 + 0.70**
9.39 + 0.24
3.00 + 0.12
a Not detectable. b Total protein phosphorylation ratio express the amount of phosphorylation in the total lane in the presence of calcium/calmodulin divided by phosphorylation in the total lane in the absence of calcium/calmodulin. * Significantly different from control (P < 0.05). ** Significantly different from control (P < 0.01). * * * Significantly less than the arithmetic average of control and ischemic value.
duced
in
ischemic
cytosolic
samples
(1.55
+
0.18;
W h e n e q u a l aliquots of c o n t r o l and i s c h e m i c l u m b a r
c o n t r o l s , 1.40 + 0.08). In t h e p r e s e n c e of C a 2 + / C a M ,
tissue
total p r o t e i n p h o s p h o r y l a t i o n in i s c h e m i c cytosolic sam-
kinase
ples (6.14 ___ 0.44) was significantly l o w e r than control
particulate
cytosol (14.98 + 1.10, P < 0.01). It is not likely that the r e d u c e d C a Z + / C a M - d e p e n d e n t
cytosolic f r a c t i o n (Table I). A similar r e s p o n s e was f o u n d
p r o t e i n kinase p h o s p h o r y l a t i o n in i s c h e m i c tissue is due
tissue (Fig.
to a l t e r e d substrate
ischemic particulate
availability, since p r o t e i n
kinase
were
combined,
activity i n c r e a s e d fraction
and
Ca2+/CaM-dependent
protein
to 4 3 %
o f c o n t r o l s in the
to 7 0 %
of c o n t r o l s
in the
in substrate p h o s p h o r y l a t i o n ratios in c o m b i n e d l u m b a r 1 and T a b l e II). C o m b i n e d samples
yielded
c o n t r o l and
substrate
phos-
activity is r e d u c e d e v e n w h e n an e x o g e n o u s substrate (10
p h o r y l a t i o n ratios that w e r e close to o r g r e a t e r t h a n the
a m i n o acid p e p t i d e ) is a d d e d to tissue samples. O t h e r possible e x p l a n a t i o n s for i m p a i r e d C a 2 + / C a M - d e p e n d e n t
a r i t h m e t i c a v e r a g e of individual s a m p l e s , e x c e p t for M r 24,000 and 31,000 p r o t e i n p h o s p h o r y l a t i o n ratios, which
p h o s p h o r y l a t i o n are r e d u c e d p r o t e i n kinase levels d u e to
w e r e closer to i s c h e m i c v a l u e s t h a n to the a r i t h m e t i c
enzyme
degradation
or
generation
of
an
inhibitory
a v e r a g e of t h e t w o s a m p l e s (Table II). All substrate
activity in i s c h e m i c tissue 13. If an inhibitory activity is
phosphorylation
p r e s e n t in i s c h e m i c tissue, the m a g n i t u d e of p h o s p h o r y -
close to c o n t r o l values. Similar results w e r e o b t a i n e d in
lation in a m i x t u r e o f e q u a l a m o u n t s of control and
total p r o t e i n p h o s p h o r y l a t i o n in p a r t i c u l a t e and cytosolic fractions. Total p r o t e i n p h o s p h o r y l a t i o n in m i x e d c o n t r o l
ischemic tissue w o u l d be less t h a n the a v e r a g e o f e a c h individual reaction. If no inhibitors w e r e p r e s e n t , and
ratios in the c o m b i n e d
cytosol w e r e
and i s c h e m i c samples was 6 5 % o f c o n t r o l (11.34 + 1.54)
a s s u m i n g no i n t e r a c t i o n s , we w o u l d e x p e c t C a 2 + / C a M -
in the p a r t i c u l a t e fraction and 8 7 % of c o n t r o l (13.00 +
d e p e n d e n t kinase activity or substrate p h o s p h o r y l a t i o n ratios (relative a m o u n t s of p h o s p h o r y l a t i o n in the prese n c e of C a 2 + / C a M d i v i d e d by p h o s p h o r y l a t i o n in the
0.50) in the cytosol. This s u g g e s t e d that no inhibitory
a b s e n c e of activators) of the m i x t u r e to b e the a r i t h m e t i c a v e r a g e of individual control and ischemic samples.
activity was p r e s e n t in i s c h e m i c tissue. T h e in v i t r o p h o s p h o r y l a t i o n p a t t e r n of e n d o g e n o u s p r o t e i n s by e n d o g e n o u s C a 2 + / C a M - d e p e n d e n t p r o t e i n kinase in the thoracic r e g i o n of the spinal c o r d was
145 TABLE III In vitro phosphorylation of endogenous substrates by endogenous Ca3+/CaM.dependent protein kinase in thoracicspinal cord
Substrate phosphorylation ratios express the relative amounts of phosphorylation in the presence of activators (calcium/calmodulin) divided by the phosphorylation in the absence of activators. Values represent means + S.E.M. (n = 4). kDa
Substrate phosphorylation ratios Particulate
Cytosol
Control
Ischemic
Control
lschemic
300 120 80 60 57 55 46 24 22 12
2.48 + 0.56 3.26 + 0.41 2.56 + 0.44 4.11 _ 0.68 8.61 + 0.05 3.51 + 1.20 2.88 + 0.04 8.89 _+ 1.00 1.91 + 0.42 4.68 + 0.53
3.84 + 0.01 3.68 + 0.95 2.25 + 0.08 4.65 + 0.55 7.50 + 0.21 2.88 + 0.06 3.85+ 0.31 8.49 ___0.12 2.23 + 0.67 3.84 + 0.01
_a 4.16 + 0.66 6.69 + 0.67 2.47 + 0.82 -
_ 3.33+ 0.16 8.65+ 0.34 1.91 +_0.25 -
Totalb
2.77 + 0.06
2.62 + 0.38
3.63+ 0.44
2.20 + 0.26
a Not detectable. b Total protein phosphorylation ratio expresses the amount of phosphorylation in the total lane in the presence of calcium/ calmodulin divided by phosphorylation in the total lane in the absence of calcium/calmodulin.
+ 2.59 in control particulate samples, and 23.02 + 2.02 in samples from ischemic animals. In thoracic cytosol, basal phosphorylation was 7.21 + 1.57 in controls, and 7.51 + 0.46 in ischemic animals; and activated phosphorylation was 17.26 + 0.79 in controls, and 16.37 + 0.88 in ischemic animals. These results show that altered Ca2+/CaM-depen dent protein kinase phosphorylation reactions are limited to the ischemic region of the spinal cord. In the present study, we found that the Ca2+/CaMdependent protein kinase phosphorylation system was impaired during irreversible ischemia in the rabbit spinal cord. Two other studies have examined the effects of ischemia on Ca2+/CaM-mediated protein phosphorylation during ischemia and recovery in the gerbil hippocampus 1'13. Both studies show that in vitro phosphorylation of endogenous substrates by endogenous Ca2+/CaM-dependent protein kinase was reduced when circulation was restored for 2 h after a 5 rain ischemic insult in adult gerbil hippocampus. This reduction in Ca2÷/CaM-dependent protein phosphorylation persisted after recirculation for 7 days. This implies that Cae+/CaM-dependent protein kinase activity may be susceptible to permanent inactivation in vivo after brief episodes of cerebral ischemia. However, it is still uncertain whether this inactivation is an initiating event in the pathogenesis of ischemic cell damage or an end result of irreversible ischemic injury. To intervene in the neuronal death caused by ischemia, it is important to understand the molecular mechanisms which render neurons unable to recover from ischemic stress. To study this process of neuronal death, it is essential to identify the biochemical reactions which are involved in the physiological changes during the critical period. Decreased Ca2÷/CaM-dependent protein kinase activity may be an important marker to follow the ischemic process. We have previously shown that the c A M P - d e p e n d e n t protein phosphorylation reactions seem to be intact in the critical phases of ischemic injury, while calcium/phospholipid-dependent protein phosphorylation is selectively impaired after irreversible CNS ischemia in the rabbit spinal cord 17. These findings suggest that aberrant calcium-dependent phosphorylation reactions are specific changes of the ischemic process, and not the result of derangement of general protein phosphorylation.
evaluated in control and ischemic rabbits to verify that r e d u c e d C a 2 + / C a M - d e p e n d e n t protein phosphorylation was limited to ischemic regions of the spinal cord. A o r t i c occlusion in ischemic rabbits does not affect thoracic spinal cord perfusion (non-ischemic). The Ca2÷/CaM d e p e n d e n t p r o t e i n p h o s p h o r y l a t i o n p a t t e r n in thoracic particulate tissue was similar to l u m b a r tissue, except the magnitude of p h o s p h o r y l a t i o n was lower in thoracic tissue (Table III). M i n o r p h o s p h o p r o t e i n s (M r 17,000, 31,000, 52,000 and 140,000) were absent in the particulate fraction from thoracic spinal cord. Cytosolic protein p h o s p h o r y l a t i o n profiles were different in thoracic and l u m b a r tissue. A d d i t i o n of Ca2+/CaM only stimulated p h o s p h o r y l a t i o n of 3 cytosolic proteins, M r 80,000, 44,000 and 21,000, in thoracic tissue. In vitro phosphorylation of e n d o g e n o u s proteins by endogenous Ca2+/ C a M - d e p e n d e n t protein kinase was similar in control and ischemic thoracic spinal cord. Basal phosphorylation was 7.48 + 1.10, n = 4, in control particulate samples, and 8.96 + 1.29, n = 4, in ischemic particulate samples. Total p h o s p h o r y l a t i o n in the presence of Ca2+/CaM was 20.66
This research was supported by research grants from the National Institutes of Health (NS 23814 and NS 28121) and the McKnight Endowment Fund for Neuroscience.
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