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Endocrinology Vol. 138, No. 7 2784-2789
Copyright © 1997 by The Endocrine Society

ARTICLES

Orthovanadate Stimulates Cyclic Guanosine Monophosphate-Inhibited Cyclic Adenosine Monophosphate Phosphodiesterase Activity in Isolated Rat Fat Pads through Activation of Particulate Myelin Basic Protein Kinase by Protein Tyrosine Kinase

Hiroshi Ueki, Shuichi Mitsugi, Yoshihito Kawashima, Toshio Motoyashiki and Tetsuo Morita

Department of Biochemistry, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshima 729–02, Japan

Address all correspondence and requests for reprints to: Hiroshi Ueki, Department of Biochemistry, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshima 729–02, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Involvement of protein kinases in the stimulation of cGMP-inhibited cAMP phosphodiesterase (PDE) activity by orthovanadate (vanadate) was studied. When the fat pads were incubated with 2 mM vanadate or 10 nM insulin, the stimulation of myelin basic protein kinase (MBPK) activity in the particulate by vanadate reached a maximum at 60 min. In contrast, insulin showed a transient increase at 20 min. A 60-min incubation of the fat pads with vanadate stimulated all activities of protein tyrosine kinase (PTK), MBPK, and PDE in the particulate, in a similar dose-dependent manner. Amiloride, a PTK inhibitor, inhibited the stimulations of three enzymes by vanadate in a similar concentration range. Enzyme fractions, which were separated from the solubilized particulate, were subjected to the immunoblot analysis. A fraction of MBPK was identified to contain a major protein of mol wt (44K) and a minor one (42K), both of which are immunoreactive with a mitogen-activated protein kinase (MAPK) antibody. The partially purified PDE activity was stimulated by the addition of the partially purified MBPK. The further stimulation was observed with the PTK-activated MBPK. These results suggest that vanadate stimulates in part the PDE activity through the activation of the particulate MBPK, probably MAPKs, by PTK sensitive to vanadate.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
S ODIUM orthovanadate (vanadate) mimics many in vitro effects of insulin including the stimulatory phosphorylation of insulin receptor, the promotion of glycogen synthesis, and the suppression of the hormone-dependent lipolysis in isolated rat adipocytes (1, 2), and an increase in lipoprotein lipase activity in rat fat pads (3). Furthermore, oral administration of vanadate normalizes blood glucose and triglyceride levels in streptozotocin-induced diabetic rats, without restoring low levels of circulating insulin (4, 5). Vanadate, however, differs from insulin in that tyrosine phosphorylation of the insulin receptor stimulated by vanadate are small and nonspecific (6). Cyclic nucleotide phosphodiesterases (PDEs), which catalyze the hydrolysis of cyclic nucleotide, are classified into five major isozyme families (7). Of these isozymes, cGMP-inhibited cAMP PDE (type III or IV in other studies) is found in the particulate of adipose tissues, and its activity is stimulated by insulin. When isolated rat fat pads were incubated with vanadate, the PDE activity in the particulate was stimulated, in a time- and dose-dependent manner (8). The stimulation was inhibited by protein tyrosine kinase (PTK) and protein kinase C (PKC) inhibitors to various extents. The PKC activity in the particulate also was stimulated by vanadate, in a time- and dose-dependent manner. These results have shown that vanadate stimulates in part the PDE activity via the activation of a PKC-mediated process. However, involvement of PTK in the stimulation of PDE is still unknown.

The present paper shows that vanadate stimulates in part the PDE activity through the activation of myelin basic protein kinase (MBPK), probably mitogen-activated protein kinases (MAPKs), by PTK sensitive to vanadat


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The sources of chemicals used in this work are as follows: Vanadate (Na3VO4) from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan); phenyl-cellulofin from Seikagaku Kogyo Co. Ltd. (Tokyo, Japan); amiloride, insulin (bovine pancreas, 27.5 IU/mg), myelin basic protein (MBP), histone II-A and III-S, and poly(Glu4,Tyr1) copolymer from Sigma Chemical Co. (St. Louis, MO); diethylaminoethyl (DEAE)-Sephacel from Pharmacia LKB Biotechnology (Uppsala, Sweden); antirat MAP kinase R2 (erk1-CT, rabbit polyclonal IgG) from Upstate Biotechnology Inc. (Lake Placid, NY); horseradish peroxidase conjugated antirabbit IgG (prepared in goat) from BioMakor (Rehovot, Israel); Renaissance from DuPont-New England Nuclear Research Products (Boston, MA); Millipore Transfer Membranes from Millipore (Bedford, MA); Bio-Rad Protein Assay kit from Nippon Bio-Rad Laboratories, K.K. (Tokyo, Japan); Aquasol-2 and [2,8-3H]cAMP (1.158 TBq/mmol) from New England Nuclear; [{gamma}-32P]ATP (167 TBq/mmol) from ICN Biomedicals, Inc. (Irvine, CA). All other chemicals used were of analytical grade.

Animals
Male Wister rats weighing 200–220 g were purchased from Hiroshima Laboratory Animals Co. (Hiroshima, Japan), maintained for 1 week in according to the Guide for the Care and Use of Laboratory Animals established by Fukuyama University, and fasted for 24 h before each experiment.

Preparation of fat pads and incubation with vanadate
Epididymal adipose tissue was quickly removed from rats killed under ether anesthesia and cut into small pieces 30–40 mg with scissors in Krebs-Ringer bicarbonate buffer, pH 7.4, containing 5 mM glucose and 2% BSA (KRBGA) at 37 C. We have reported that the stimulatory effect of vanadate on the PDE activity was not different between the fat pads and adipocytes (8). Therefore, the fat pads were used through this experiment. The stock solution of vanadate (100 mM, pH 7.4) was diluted with deionized water to the desired concentration immediately before use. The fat pads (1 g) were incubated with vanadate in 10 ml of KRBGA at 37 C for 60 min, washed three times with physiological saline at 4 C, and used as materials of next step.

Preparation of subcellular fractions
Subcellular fractions were prepared from the incubated fat pads, as described previously (8, 9). Briefly, the incubated fat pads (1 g) were homogenized with a Potter-Elvejehm homogenizer in 2 ml of 10 mM Tris-HCl buffer, pH 7.4, containing 0.25 M sucrose and protease inhibitors (1 mM benzamidine and 0.1 mM p-amidino-phenylmethanesulfonyl fluoride). After centrifugation at 1,000 x g for 10 min, the infranatant was further centrifuged at 12,000 and 17,500 x g for 4.5 min each to remove a mitochondrial fraction and at 105,000 x g for 60 min to separate a particulate fraction. The particulate was used as enzyme sources through this experiment.

Column chromatography
The particulate (12 mg protein) was solubilized in 1 ml of 0.5% Triton X-100 containing 5 mM 2-mercaptoethanol and the protease inhibitors at 4 C for 1 h and centrifuged at 13,000 x g for 10 min. The resultant supernatant (1 ml) was applied to a DEAE-Sephacel column (7.5 ml) previously equilibrated with 10 mM Tris-HCl buffer, pH 7.0, containing 30 mM 2-mercaptoethanol, 0.1 mM EDTA, 10% glycerol, and the protease inhibitors (9, 10, 11). The buffer, hereafter, is referred to as a column buffer. The column was washed with 40 ml of the column buffer and eluted stepwise with 0.2–0.4 M NaCl in the column buffer. For the further purification, fraction nos. 19–21, containing PTK and MBPK activities, (1 ml) were applied to a phenyl-cellulofin column (2 ml) previously equilibrated with the column buffer (12). After washing with 10 ml of the column buffer, elution was carried out with a gradient of 0–40% ethylene glycol in the column buffer. Protein concentration was calculated from the absorbance at 280 nm or determined using a Bio-Rad protein assay kit.

Determination of PDE activity
The PDE activity was determined by a slight modification of the method of Kono et al. (8, 13). An aliquot (0.05 ml) of enzyme solution was incubated with 250 nM [3H] cAMP (0.9 KBq) in 33 mM Tris-HCl buffer, pH 7.4, containing 4 mM MgCl2 at 30 C for 20 min, in a total volume of 0.25 ml. The incubation was terminated by the addition of 0.1 ml of 0.1 N HCl. A mixture (0.05 ml) of 5 mM AMP and cAMP was added to the incubation mixture, allowed to stand at 70 C for 4 min, cooled, and neutralized with 0.1 ml of 0.1 N NaOH. After the addition of 50 µg snake venom in 0.05 ml of 0.1 M Tris-HCl buffer, pH 8.0, to the neutralized solution, the incubation was carried out at 37 C for 20 min and terminated by the addition of a mixed solution (0.05 ml, pH 7.0) of 200 mM EDTA and 5 mM adenosine. The reaction mixture (0.5 ml) was applied to a 5.5 x 30 mm column of Dowex 1-X8 (200–400 mesh in chloride form). The loaded column was eluted with water. The first 1.4 ml of the effluent was discarded, and the next 2.5 ml was collected. The radioactivity of adenosine contained in the 2.5 ml fraction was determined in Aquasol-2 with an Aloka liquid scintillation counter (LSC-700). The PDE activity was expressed in terms of picomoles of cAMP hydrolyzed per min per mg protein.

Determination of MBPK activity
The MBPK activity was determined by a modification of the method of Meier et al. (14). A mixture (0.1 ml) of enzyme solutions (0.04 ml) and 20 mM HEPES buffer, pH 7.5, containing 10 mM MgCl2, 2 mM DTT, 50 µM [32P]ATP (3.3 KBq), 0.02% Triton X-100, and 33 µg MBP was incubated at 30 C for 15 min. The reaction was terminated by adding 2 ml of 5% trichloroacetic acid (TCA) containing 0.025% sodium tungstate. After adding 0.05 ml of 1.3% BSA and standing at 4 C for 5 min, it was centrifuged at 13,000 x g for 10 min. The precipitate obtained was dissolved in 0.05 ml of 1 N NaOH, to this added 1 ml of the TCA solution and 0.05 ml of 1.2 N H2SO4, and centrifuged. This procedure was repeated once again. The precipitate was finally dissolved in 0.5 ml water, and its radioactivity was determined. The MBPK activity was expressed in terms of picomoles of 32P incorporated into the substrates per min per mg protein.

Determination of PTK activity
For the determination of the PTK activity, a mixture of 0.02 ml of enzyme solutions and 0.04 ml of 20 mM HEPES buffer, pH 7.4, containing 15 mM MgCl2, 2 mM MnCl2, 0.2% Triton X-100, 0.1 mg poly(Glu4,Tyr1) copolymer, and 1 µM [32P]ATP (11, 7 KBq), was incubated at 25 C for 10 min (9, 15, 16). The reaction was stopped by the addition of 1 mM ATP. After centrifugation at 13,000 x g for 3 min, the supernatant containing phosphorylated substrate was applied to Whatman 3MM paper squares (2 x 2 cm) and washed with 10 and then 5% TCA, containing 2 mM sodium pyrophosphate. The paper strips were dried and analyzed for their radioactive contents. The PTK activity was expressed in terms of picomoles of 32P incorporated into the substrates per min per mg protein.

Gel electrophoresis and Western blot analysis
Samples were subjected to SDS-PAGE with 9% slab gels (17) and thereafter immunoblot procedures, essentially according to the method of Towbin et al. (18). Briefly, proteins in the gels were transferred electrophoretically to polyvinylidene difluoride membranes (Millipore Transfer Membranes). The membranes were treated with 0.01 M phosphate buffer, pH 7.2, containing 0.15 M NaCl and 10% skim milk, then probed with the antirat MAPK antibody that recognizes the mol wt 42K, 43K, and 44K MAPKs, respectively. After incubation of the probed membranes with the horseradish peroxidase conjugated antirabbit IgG, the positive bands were visualized by using Western blot chemiluminescence reagent (Renaissance) and exposed to Kodak diagnostic film.

Statistical analysis
All results are presented as mean ± SE of four observations. Similar results were obtained with at least two separate experiments. The data were analyzed by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of vanadate on MBPK, PTK, and PDE activities
Figure 1Go shows changes in the MBPK activity in the fat pads incubated with 2 mM vanadate or 10 nM insulin up to 150 min. The stimulation of MBPK activity by vanadate reached a maximum with a 60-min incubation period and was sustained for 150 min. In contrast, insulin showed a transient increase in the MBPK activity with a 20-min incubation period and then a rapid decrease to the original level. The stimulatory profile of vanadate was similar to the time course of stimulation of the MBPK activity in adipocytes by okadaic acid (19). When the fat pads were incubated for 60 min with vanadate over the concentration range of 0–2 mM, PTK, MBPK, and PDE activities in the particulate were increased in a similar dose-dependent manner up to 1 mM (Fig. 2Go). These increases all were inhibited by the pretreatment of the fat pads with amiloride, a PTK inhibitor (20, 21), under the condition of no change in the basal activity of each enzyme. The concentrations required for half-maximal inhibition of the vanadate-stimulated activity by amiloride were calculated to be 1.18, 1.35, and 1.45 mM for MBPK, PTK, and PDE, respectively. These results suggest that the PTK activity sensitive to vanadate links to the stimulations of MBPK and PDE activities.



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  		Figure 1. Time course of stimulatory effect of vanadate or insulin on MBPK activity. The fat pads (1 g) were incubated with 2 mM vanadate or 10 nM insulin or without either one in 10 ml of KRBGA for 0–150 min, homogenized, and centrifuged as described in Materials and Methods. The resultant particulates were assayed for MBPK activity.

 


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  		Figure 2. Dose-response curves for stimulatory effect of vanadate on PTK, MBPK, or PDE activity. The fat pads (1 g) were incubated with vanadate at the indicated concentrations for 60 min, homogenized, and centrifuged as described in Materials and Methods. The resultant particulates were assayed for activities of PTK (A), MBPK (B), and PDE (C). *, P < 0.05; **, P < 0.01, compared with groups without vanadate.

 
Partial purification of PTK, MBPK, and PDE
We next attempted to separate PTK, MBPK, and PDE activities. The supernatant of solubilized particulate was applied to a DEAE-Sephacel column, then eluted stepwise with 0.2, 0.3, and 0.4 M NaCl in the column buffer (Fig. 3Go). Fraction no. 7 (DS 7) was eluted with 0.2 M NaCl in the column buffer, as first peak of MBPK activity. This did not show any positive protein bands, which are immunoreactive with MAPK antibody, by immunoblot analysis, as described later. Fraction nos. 19–21 (DS 19–21), which were eluted with 0.3 M NaCl in the column buffer, were used as the samples of further purification of MBPK and PTK activities. The fraction no.35 (DS 35) was eluted with 0.4 M NaCl in the column buffer from the column, as a single peak of PDE activity. For the further purification, DS 19–21 was applied to a phenyl-cellulofin column and eluted with a gradient of increasing concentration of 0–40% ethylene glycol in the column buffer (Fig. 4Go). Fraction no. 17 (PC 17) and 24 (PC 24) were used as fractions containing PTK and MBPK activities, respectively, due to relatively low contamination. A partially purified PDE (DS 35) showed neither activity of MBPK nor PTK. A partially purified PTK (PC 17) contained a high PTK activity and a slight MBPK activity. A partially purified MBPK (PC 24) showed MBPK activity alone. The phosphorylation activities of the partially purified MBPK against proteins known to be substrates for other protein kinases were tested (14, 15, 22, 23). MBP was preferentially phosphorylated. Phosphorylation of histone III-S was only 0.2% of that of MBP. No phosphorylation was observed with substrates such as histone II-A, poly(Glu4,Tyr1) copolymer, and casein.



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  		Figure 3. DEAE-Sephacel chromatography of PTK, MBPK, and PDE. An aliquot (10 mg protein) of the solubilized particulate was applied to a DEAE-Sephacel column (7.5 ml) previously equilibrated with the column buffer. After washing the column with the column buffer, elution was carried out stepwise with 0.2, 0.3, and 0.4 M NaCl in the column buffer, as shown with the arrow. Fractions (2 ml) were collected and assayed for PTK, MBPK, and PDE activities.

 


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  		Figure 4. Phenyl-cellulofin chromatography of PTK and MBPK. DS 19–21 containing PTK and MBPK activities (1 ml) was applied to a phenyl-cellulofin column (2 ml) previously equilibrated with the column buffer. After washing the column with the column buffer, elution was carried out with a gradient of increasing concentration of 0–40% ethylene glycol in the column buffer. Fractions (1 ml) were collected and assayed for PTK and MBPK activities.

 
Identification of MAPK-like proteins
Figure 5Go shows inhibitory profiles of two MAPK inhibitors (12). The MBPK activity in the particulate was decreased approximately 50% with 40 mM potassium fluoride and completely lost at the 120 mM concentration. A weak inhibition was observed with ß-glycerophosphate even at a high concentration of 160 mM. Immunoblot analysis indicated that the solubilized particulate and DS 19–21 contained a major protein of mol wt 44K and a minor one of 42K, both of which were immunoreactive with MAPK antibody (Fig. 6Go). In contrast, the immunoreactive protein bands were barely visible with DS 7 and DS 35.



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  		Figure 5. Effects of potassium fluoride and ß-glycerophosphate on MBPK activity in particulate. The particulate (10 µg protein) was incubated with inhibitors at the indicated concentrations for 15 min and assayed for MBPK activity. Results are expressed as percentage of the control (group without inhibitors).

 


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  		Figure 6. Identification of MAPK-like proteins by Western blot analysis. Samples (15 µg protein) of the solubilized particulate (a), DS 7 (b), DS 19–21 (c), and DS 35 (d) were subjected to SDS-PAGE and thereafter immunoblot analysis using the MAPK antibody. Approximate mol wts ( x 10-3) are indicated on the left.

 
Effect of MBPK and/or PTK on PDE activity
Figure 7Go shows the effect of the partially purified MBPK or PTK alone or either one on the partially purified PDE in the presence or absence of amiloride. The PDE activity was increased 2.3-fold by the addition of the PTK-stimulated MBPK. Under this condition, the PTK increased 2.0-fold the MBPK activity (data not shown). The addition of the PTK or MBPK alone showed 1.1- or 1.5-fold increase in the PDE activity. The additive effect of the PTK on the MBPK-stimulated PDE activity was completely inhibited in the presence of 2 mM amiloride. These results suggest that the PDE activity is in part stimulated by the MBPK sensitive to the PTK.



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  		Figure 7. Effect of PTK-stimulated MBPK on PDE activity. The partially purified PDE (1.6 µg protein) was incubated with the partially purified MBPK (0.05 µg protein), which was pretreated with the partially purified PTK (0.1 µg protein) in the column buffer with or without 2 mM amiloride at 30 C for 5 min, and assayed. Results are expressed as percentage of the control (group without the PTK and MBPK).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results presented here show that the stimulation of the PDE activity in the fat pads by vanadate is due to the activation of MBPK, probably MAPKs, by PTK sensitive to vanadate. Stimulation of 3T3-L1 cells by insulin has been reported to activate a cytosolic serine/threonine kinase capable of phosphorylating microtubule-associated protein 2 (12, 24, 25). This enzyme has been termed MAPK. In an insulin-stimulated phosphorylation cascade, MAPK was activated by dual phosphorylation at tyrosyl and threonyl residues of the enzyme (26). It is unclear, however, whether or not the stimulatory phosphorylation cascade via MAPKs are involved in the regulation of the particulate PDE activity by insulin. The insulin-activated MBPK in rat adipocytes is referred to as MAPK and most of mammalian MAPKs are isoforms of mol wt 42K and 44K, also referred to as ERK 2 and ERK 1, respectively (27, 28). A recent report has shown that vanadate stimulates tyrosine phosphorylation of two major proteins with molecular masses of 42K and 44K in Chinese hamster ovary cells overexpressing a normal human insulin receptor, and that the stimulatory tyrosine phosphorylation is associated with an activation of MAPK (29). Although mechanisms of the vanadate action have been not clearly shown, a possible explanation has been that vanadate increases the phosphotyrosyl content of MAPK by preventing the dephosphorylation, due to a potent inhibitor of protein tyrosine phosphatases (30, 31). In contrast, our results were that PTK, MBPK, and PDE activities all were stimulated by vanadate and that these stimulations were inhibited by amiloride to a similar extent, suggesting the linkage of the PTK and MBPK to the stimulation of the PDE activity.

To confirm the activation of PDE by PTK and MBPK, each enzyme was partially purified by column chromatography. DS 19–21 was identified to contain a major protein of mol wt 44K and a minor one of 42K by immunoblotting with a specific antibody to MAPKs, suggesting that most of MBPK activity in DS 19–21 are indeed attributed to MAPKs, mainly ERK 1. The addition of the partially purified MBPK (PC 24) to the partially purified PDE (DS 35) showed a distinct increase in the PDE activity. Further increase was observed by the addition of the PTK-stimulated MBPK. In contrast to DS 19–21, DS 7, which did not contain the immunoreactive proteins, never showed the stimulatory effect on the PDE activity (data not shown). These results show that the MBPK, containing proteins that are immunoreactive with MAPK antibody, stimulates the PDE activity, and that the PTK sensitive to vanadate is a major upstream activator for the MBPK to stimulate the phosphorylation for the PDE activation cascade. The partially purified MBPK seems to be probably identical to the MAPKs that are activated by the incubation of the fat pads with vanadate. We have not yet found that the particulate PTK activity is stimulated by the incubation of the fat pads with insulin. Therefore, identification of MBPK activated by insulin and involvement of it in the activation of PDE are under study at the present time.

The cytosolic MBPK activator, which has been partially purified from rat liver, stimulates MBPK activity in vitro in the presence of Mg2+ and ATP, concomitantly with phosphate incorporation into both tyrosyl and threonyl residues of MBPK (32). The cytosolic PTK is activated by vanadate in rat adipocytes and shows an optimal divalent ion requirement of 15 mM Mg2+ + 2 mM Co2+ in the assay medium (16). In contrast, the optimal divalent ion requirement for the particulate PTK activity was Mn2+ rather than Co2+, in addition to Mg2+ (9). N-Ethylmaleimide (1 mM) inactivated the insulin receptor PTK activity but did not inactivated the particulate PTK activity (9, 16). Thus, the particulate PTK appears to have some properties different from PTKs in cytosol and insulin receptor. It is unknown, at the present time, whether the cytosolic PTK or MBPK activator is involved in the PDE activation cascade.

The insulin-stimulated PDEs purified from the particulate fraction of rat adipose tissue and adipocytes have been reported as mol wt 44K–135K proteins that are immuno-reactive with PDE antibody (33, 34, 35). Finally, the mol wt calculated from the amino acid sequence encoded by complementary DNA (cDNA) for the insulin-stimulated PDE of the rat adipocyte has been reported to be 123,091 (36). Furthermore, it has been reported that other PDE isoforms that are products of different genes may exist in vascular element of rat adipose tissue (36). Whether vanadate stimulates the insulin-stimulated PDE itself or other isoforms remains to be elucidated. Serine 427 in the rat adipocyte PDE of mol wt 135K is phosphorylated by cAMP-dependent protein kinase (PKA) in vitro (37). Vanadate shows a rapid increase in the cAMP content accompanied by the activation of PKA in the fat pads (38). There is, therefore, the possibility that PKA may be involved in the stimulation of the PDE by vanadate. The model in Fig. 8Go illustrates that vanadate may activate PTK, PKC, and PKA through signals from various types of cell surface receptors to stimulate the PDE activity. The vanadate-stimulated PTK shows the sequential stimulation of the MAPK and PDE activities. The vanadate-stimulated PKC could stimulate the PDE activity through the activation cascade involving Ras, Raf, MAPK kinase (MAPKK), and MAPK (8, 39). The vanadate-stimulated PKA might stimulate the PDE activity by a direct phosphorylation of a specific serine in the PDE (37).



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  		Figure 8. Proposal mechanism of action of vanadate on stimulation of PDE activity in rat fat pads. Solid arrows indicate the sequential stimulation of protein kinase and PDE activities. Question marks represent unknown steps or possible activation processes.

 
In conclusion, vanadate seems to stimulate in part the PDE activity in the particulate through a process involving the activation of MBPK, probably MAPKs, by PTK sensitive to vanadate, in addition to an activation cascade involving the PKC.

Received December 6, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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