Effects of riluzole on N-methyl-D-aspartate-induced tyrosine phosphorylation in the rat hippocampus

A hydroxysteroid sulfotransferase (ST2A1) was identified as a form mediating neurosteroid sulfation in rat brain. The sole expression among known rat ST2A forms was indicated by brain RT-PCR. All nucleotide sequences of seven ST2A cDNA clones isolated from brain matched completely with that of hepatic ST2A1. The recombinant ST2A1 protein mediated neurosteroid sulfation. These data strongly suggest a functional role of ST2A1 as a neurosteroid sulfotransferase in rat brain.     

Anesthetic concentrations of riluzole inhibit neuronal nitric oxide synthase activity, but not expression, in the rat hippocampus

We hypothesized that anesthetic dose of riluzole, an inhibitor of glutamate neurotransmission, may affect the activity and/or expression of neuronal NOS (nNOS). Riluzole, N(G)-nitro-L-arginine-methyl ester (L-NAME) and 7-nitro indazole (7-NI) produced a concentration-related inhibition of nNOS activity in vitro. Riluzole competed with 7-NI for inhibition of nNOS activity, but had no effect on nNOS or endothelial NOS (eNOS) protein expression. Also, nNOS activity was significantly decreased in riluzole-anesthetized rats (40 mg kg(-1) i.p., -32+/-6% from controls, P<0.05). Therefore, blockade of nNOS activity may be involved in the anesthetic effects of riluzole in vivo.     

Calcium-, calcium/calmodulin-, and calcium/phospholipid-stimulated protein phosphorylation in the rat anterior pituitary.

Calcium-dependent protein phosphorylation may be a critical step in the stimulated secretion of anterior pituitary hormones. We have noted the existence of a number of calcium-calcium/calmodulin-, and calcium/phospholipid-dependent phosphoproteins in the normal rat anterior pituitary. Cell extracts were prepared from anterior pituitary glands of male rats and phosphorylated with [gamma 32P]ATP in the presence or absence of calcium, calmodulin, and phosphatidylserine. The samples were electrophoresed on SDS-PAGE gels, autoradiographs prepared, and phosphate incorporation into specific proteins quantitated with microdensitometry. Calcium alone significantly stimulated the phosphorylation of proteins with molecular weights of 80.0-, 62.0-, 51.0-, 30.5-, and 25.0-kDa. The phosphorylation of 21.5-, 51.0-, and 80.0-kDa MW phosphoproteins was found to be phospholipid dependent. The phosphorylation of 62.0-, 51.0-, 33.0-, 30.5-, and 25.0-kDa MW phosphoproteins was found to be calcium/calmodulin kinase dependent. Calcium/calmodulin also inhibited phosphorylation of the 80.0-kDa phosphoprotein.     

Chronic lithium administration alters a prominent PKC substrate in rat hippocampus

The therapeutic effect of lithium in the treatment of bipolar disorder exhibits a significant delay in the onset of action and a persistence of efficacy beyond abrupt discontinuation of treatment. Lithium is known to alter receptor-coupled phosphoinositide second messenger pathway in brain, resulting in indirect changes in an endogenous activator of protein kinase C (PKC). Such evidence has suggested that PKC may be involved in the mechanism of action of lithium in the brain. PKC represents a site wherein long-term regulatory changes in cell function occur through the phosphorylation of specific phosphoproteins involved in processes including neurotransmitter release and receptor activation. In studies of rats exposed to lithium, however, we have found no significant effects of chronic administration on the relative activity, subcellular distribution, or activation of PKC in hippocampus. We did find a major reduction in the in vitro PKC mediated phosphorylation of two major substrates, 83 kDa and 45 kDa, in hippocampus of rats exposed to chronic lithium and maintaining clinically relevant therapeutic levels in brain. Using immunoblot analysis we have identified a known myristoylated alanine-rich C kinase substrate (MARCKS) at 83 kDa. In vivo levels of MARCKS in hippocampus were found to be significantly reduced after chronic lithium exposure. These findings persist in animals withdrawn from lithium, but are not apparent following acute treatment. In light of the potential role of PKC substrates such as MARCKS in signal transduction and the fact that there appear to be changes in the intracellular concentration of MARCKS that parallel the time course of clinical action of lithium, we suggest the possible involvement of these proteins in its mechanism of action in the treatment of bipolar disorder.     

Hyperglycemia and hypercapnia differently affect post-ischemic changes in protein kinases and protein phosphorylation in the rat cingulate cortex

Hyperglycemia and hypercapnia aggravate intra-ischemic acidosis and subsequent brain damage. However, hyperglycemia causes more extensive post-ischemic damage than hypercapnia, particularly in the cingulate cortex. We investigated the changes in the subcellular distribution of protein kinase Cgamma (PKCgamma) and the Ca2+/calmodulin-dependent protein kinase II (CaMKII), as well as changes in protein tyrosine phosphorylation during and following 10 min normoglycemic, hyperglycemic (plasma glucose approximately 20 mM) and hypercapnic (paCO2) approximately 300 mm Hg) global cerebral ischemia. During reperfusion period, the translocation to cell membranes of PKCgamma, but not CaMKII, was prolonged by intra-ischemic hyperglycemia, while it was only marginally affected by hypercapnia. The tyrosine-phosphorylation of proteins in the synaptosomal membranes, as well as the extracellular signal-regulated kinase (ERK) in the cytosol, markedly increased during reperfusion following hyperglycemic ischemia, but to a lesser degree following hypercapnic ischemia. Our data suggest that PKCgamma, tyrosine kinase and ERK systems are involved in the process of ischemic damage in the cingulate cortex, where hyperglycemia may affect these kinases through an additional mechanism other than exaggerated acidosis.     

Cyclic AMP-dependent protein phosphorylation in the rat anterior pituitary

The activation of cyclic adenosine 3'5'-monophosphate (cAMP)-dependent protein kinases has been implicated as an integral mechanism in stimulus-secretion coupling in the anterior pituitary. Therefore, we have investigated phosphorylation of endogenous protein substrates both in the presence and absence of cAMP in cell-free extracts of the rodent anterior pituitary. Specific phosphoprotein substrates in the rat anterior pituitary, which are phosphorylated by a cAMP-dependent protein kinase in vitro, were identified. Cyclic AMP potentiated the phosphorylation of proteins with apparent molecular weights of 85,000, 77,000, 63,000, 53,000, 39,000, and 33,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins with apparent molecular weights of 124,000, 93,000, 48,000, and 43,000 were phosphorylated only in the presence of cAMP and not in the basal condition. The results highlight endogenous protein substrates that may potentially be involved in cAMP-dependent stimulus-secretion coupling in the anterior pituitary.     

Long-lasting increase in protein kinase C activity in the hippocampus of amygdala-kindled rat

Previous studies have demonstrated that membrane-associated protein kinase C (PKC) activities in the right and left hippocampus of rats kindled from the left hippocampus increased significantly at 4 weeks [9] and 4 months [22] after the last seizure compared with those in matched control rats. In this study, we investigated the effect of kindling from the left amygdala on PKC activities in the amygdala/pyriform cortex and hippocampus at long seizure-free intervals (4 and 16 weeks) from the last amygdala-kindled seizure. Membrane-associated PKC activity of the kindled group increased significantly only in the left hippocampus compared with the left side control (the left hippocampus of rats subjected to a sham operation) at 4 weeks (by 34%, P < 0.03) and 16 weeks (by 24%, P < 0.05) after the last seizure. There was no significant alteration in the membrane-associated PKC activity of the kindled group in the right hippocampus or amygdala/pyriform cortex in any seizure-free interval after the last amygdala seizure. Cytosolic PKC activity did not differ between the kindled and control groups in any brain region examined in any seizure-free interval. At 16 weeks after the last seizure, the PKC activity in the P₁ fraction of the kindled group increased significantly only in the left hippocampus (by 49%, P < 0.005), but not in the right hippocampus. Neither PKC activity in the P₂ fraction nor that in the cytosolic fraction was altered in the kindled group after this seizure-free interval. The prolonged increase in activity of the membrane-associated PKC and that in the P₁ fraction in the hippocampus induced by amygdala-kindling may contribute to long-lasting seizure susceptibility induced by kindling.     

Effects of trifluoperazine on synaptically evoked potentials and membrane properties of CA1 pyramidal neurons of rat hippocampus in situ and in vitro

The effects of trifluoperazine (TFP), a phenothiazine antipsychotic, on hippocampal activity were studied in the CA1 subfield, both in situ and in slices. In the extracellular studies in situ and in vitro, both somatic population spikes and dendritic excitatory postsynaptic potentials (EPSP) fields were depressed reversibly by TFP, applied by microiontophoresis or in the bath (50-100 μM). Similar effects were also seen during iontophoretic applications of sphingosine in situ. Like TFP (at micromolar concentrations) sphingosine is a dual Ca²⁺/calmodulin-dependent kinase and protein kinase C (PKC) inhibitor. In intracellular recordings from slices, 50-100 μM TFP induced a slow depolarization and a decrease in input resistance (R_N), probably through a β-aminobutyric acid (GABA)-mediated increase in Cl^? conductance (G_Cl). TFP also reduced the slow afterhyperpolarization (AHP) as well as electrically evoked inhibitory postsynaptic potentials (IPSPs), but EPSPs were augmented in both amplitude and duration. When CA1 neurons were voltage clamped, TFP elicited a corresponding inward current (consistent with depolarization), increased the leak conductance, and enhanced excitatory synaptic currents; whereas inhibitory synaptic currents and high-threshold Ca²⁺ currents were reduced. In conclusion, these effects of TFP–which cannot be readily explained by its potent antidopamine action–are in keeping with other evidence that both Ca²⁺/calmodulin-dependent kinase and PKC can modulate G_Cl-conductance and high-threshold Ca²⁺ -conductance, as well as inhibitory and excitatory postsynaptic currents. © 1993 Wiley-Liss, Inc.     

Dual effects of interleukin-1β on N-methyl- -aspartate-induced retinal neuronal death in rat eyes

In this study we determine if interleukin-1β (IL-1β) modulates N-methyl- -aspartate (NMDA)-induced retinal damage. Sprague–Dawley rats were anesthetized with inhalation of halothane, after which a single injection of 5 μl of IL-1β (0.1 to 10 ng/eye) (and/or IL-1 receptor antagonist (IL-1ra)) for experimental eyes was administered. Two days later (or simultaneously), NMDA (20 nmol) was injected into the vitreous space. One week later, each eye was enucleated and transverse sections were subjected to morphometric analysis. Enzyme-linked immunosorbent assay (ELISA) was conducted for the determination of IL-1β levels in retina. Immunohistochemical and immunoblot studies were also performed. In eyes that received an intravitreal injection of IL-1β (0.1 to 10 ng/eye), significant thinning of the inner plexiform layer (IPL) was observed (P<0.05). Immunohistochemical and ELISA studies demonstrated upregulated expression of IL-1β in retinas that had undergone NMDA injection. Treatment with 10 ng of IL-1ra induced a protective effect against NMDA-induced retinal damage. Pretreatment with IL-1β induced a significant protective effect on NMDA-induced retinal damage. Our studies suggest that IL-1β induces neuronal cell death directly, as shown by the protective effects of IL-1ra, but has a protective effect on NMDA-induced retinal damage indirectly after an incubation time of at least 2 days.     

Increased tyrosine phosphorylation of alpha(1C) subunits of L-type voltage-gated calcium channels and interactions among Src/Fyn,PSD-95 and alpha(1C) in rat hippocampus after transient brain ischemia

It has been reported that the Src family kinases-mediated tyrosine phosphorylation of alpha(1C) subunits of L-type voltage-gated calcium channels (L-VGCCs) potentiates the channel currents. In this study, we evaluated the alterations in the tyrosine phosphorylation level of alpha(1C) and in the interactions involving Src/Fyn, alpha(1C) and PSD-95 in the hippocampus after transient (15 min) brain ischemia followed by various times of reperfusion using immunoprecipitation and immunoblotting. Transient brain ischemia was induced by the method of four-vessel occlusion in Sprague-Dawley rats. The tyrosine phosphorylation level of alpha(1C) subunits elevated immediately after brain ischemia. The elevation in phosphorylation sustained for at least 6 h and peaked at 15 min of reperfusion. Transient brain ischemia and reperfusion also caused rapid and sustained increases in the interactions of Src and Fyn with alpha(1C) subunits. More interestingly, co-immunoprecipitation analysis showed that 15 min of reperfusion dramatically increased the interaction between PSD-95 and alpha(1C) and promoted the formation of alpha(1C)-PSD-95-Src complexes, for the first time. The protein levels of alpha(1C), Src, Fyn and PSD-95 showed no differences at all time points. These results suggest a novel mechanism involving the ischemia/reperfusion-induced recruitment of L-VGCCs, Src and Fyn to the PSD-95 signaling complex that facilitates the tyrosine phosphorylation of alpha(1C) subunits by Src family kinases and may contribute to the up-regulation of L-VGCCs activity in postischemic hippocampus.     

Increased phosphorylation of neurofilament subunits in PC12 cells and rat dorsal root ganglion neurons treated with N-Acetyl-Leu-Leu-norleucinal

Treatment of PC12 cells or dorsal root ganglion neurons with the protease inhibitor, N-Acetyl-Leu-Leu-norleucinal, stimulated phosphorylation of the mid-sized and heavy neurofilament subunits and caused the heavy subunit in the perikarya of dorsal root ganglion neurons to become hyperphosphorylated. The closely related inhibitor, N-Acetyl-Leu-Leu-methioninal, did not produce a similar effect. Okadaic acid increased the phosphorylation state of the heavy neurofilament subunit in PC12 cells in a fashion similar to N-Acetyl-Leu-Leu-norleucinal and the effect of both compounds together was greater than for either one alone. There was no increase in cyclin-dependent kinase 5-immunoprecitable histone H1 kinase activity in PC12 cells treated with N-Acetyl-Leu-Leu-norleucinal despite the presence of enzyme protein.The present study demonstrates that a protease inhibitor can induce the hyperphosphorylation of neurofilament subunits to a level normally seen only in axons. This suggests that perturbations in intracellular proteolysis may lead to the accumulation of phosphorylated neurofilament epitopes in neuronal perikarya in certain pathological states. The results also show that the carboxy-terminal tail domains of the two largest neurofilament subunits are phosphorylated even when cyclin dependent kinase 5 is inactive, indicating that other neuronal kinases are involved in the phosphorylation of Lys-Ser-Pro repeats.     

Modulation of baroreflex sensitivity by the state of protein tyrosine phosphorylation in the brainstem of the rat

Evidence accumulated recently suggests that protein tyrosine phosphorylation may play an important role in regulating neuronal functions. In the present study, we investigated if the state of protein tyrosine phosphorylation in the brainstem regulates baroreflex sensitivity. Anti-phosphotyrosine immunoblots of brainstem tissue revealed that several phosphotyrosine-containing proteins were present in the brainstem and their level of tyrosine phosphorylation was decreased by treatment of the slices with the protein tyrosine kinase (PTK) inhibitor genistein, and increased by treatment with the protein tyrosine phosphatase (PTP) inhibitor pervanadate. In urethane-anaesthetized rats, we found that inhibiting PTK activity by topical application of genistein to the dorsal surface of the medulla reduced the phenylephrine-induced baroreflex bradycardiac response. Conversely, the baroreflex response was potentiated by activating endogenous PTK activity with insulin or by inhibiting PTP activity with pervanadate. Thus these results suggest that the state of cellular tyrosine phosphorylation within the dorsal medulla of the brainstem may regulate the baroreflex control of heart rate, thereby providing the first evidence for a role for protein tyrosine phosphorylation, a key process involved in diverse intracellular signalling pathways, in modulating baroreflex sensitivity.     

Protein kinase C in the hippocampus is altered by spatial but not cued discriminations: A component task analysis

The exact role of the mammalian hippocampus in memory formation remains essentially as an unanswered question for cognitive neuroscience. Experiments with humans and with animals indicate that some types of mnemonic associative processes involve hippocampal function while others do not. Support for the spatial processing hypothesis of hippocampal function has stemmed from the impaired performance of rats with hippocampal lesions in tasks that require spatial discriminations, but not cued discriminations. Previous procedures, however, have confounded the interpretation of spatial versus cued discrimination learning with the number and kinds of irrelevant stimuli present in the discrimination.An empirical set of data describing a role of protein kinase C (PKC) in different mnemonic processes is similarly being developed. Recent work has implicated the activation of this serine-threonine kinase in a variety of learning paradigms, as well as long-term potentiation (LTP), a model system for synaptic plasticity which may subserve some types of learning.The present study employs the principles of component task analysis to examine the role of membrane-associated PKC (mPKC) in hippocampal-dependent memory when all factors other than the type of learning were equivalent. The results indicate that hippocampal mPKC is altered by performance in hippocampally-dependent spatial discriminations, but not hippocampally-independent cued discriminations and provide a general experimental procedure to relate neural changes to specific behavioral changes.     

Suppression of protein kinase C is associated with inhibition of PYK2 tyrosine phosphorylation and enhancement of PYK2 interaction with Src in thrombin-activated platelets

Blood platelets have recently been shown to express PYK2, a nonreceptor tyrosine kinase belonging to the FAK gene family. In this study, we examined the involvement of protein kinase C (PKC) in PYK2-related responses in human platelets. While PYK2 tyrosine phosphorylation induced by thrombin was inhibited by preincubation of platelets with PKC inhibitors, staurosporine and Ro31-8220, PYK2 association with Src was markedly enhanced under the same conditions. Platelet intracellular Ca²⁺ mobilization induced by thrombin was hardly inhibited by these PKC inhibitors. p130^Cas is a docking protein that associates with FAK or PYK2 through the SH3 domain. Although we identified p130^Cas in platelets for the first time, this docking protein failed to interact with PYK2. These results suggest that PKC activation (but not Ca²⁺ mobilization) is involved in PYK2 tyrosine phosphorylation and that PYK2 associates with Src without PYK2 tyrosine phosphorylation or p130^Cas involvement in platelets.     

Biphasic modulatory action of the selective sigma receptor ligand SR 31742A on N-methyl- -aspartate-induced neuronal responses in the frontal cortex

The technique of intracellular recording was used to assess the effect of SR 31742A, a selective sigma receptor ligand, on N-methyl- -aspartate (NMDA) and (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated responses in pyramidal cells of the rat medial prefrontal cortex in vitro brain slice preparations. Bath application of SR 31742A produced a biphasic effect on NMDA responses: SR 31742A facilitated and inhibited NMDA-induced inward current at low (0.01, 0.05 and 0.1 μM) and higher (0.5, 1 and 10 μM) concentrations, respectively. The potentiating effect reached the peak (366%) at 0.1 μM, with an estimated EC₅₀ value of 23 nM. Correspondingly, SR 31742A also produced a similar biphasic modulatory action on excitatory postsynaptic potentials or currents (EPSPs/EPSCs) evoked by electrical stimulation of the forceps minor. In contrast, SR 31742A produced a modest potentiation of AMPA responses at the concentrations from 0.01 to 1 μM. The potentiating action of SR 31742A on NMDA-receptor mediated neurotransmission may account for, at least partially, its antipsychotic and cognitive-enhancing potential, whereas the inhibitory action on NMDA responses at higher concentrations may be related to the purported neuroprotective action of sigma receptor ligands.     

Activators of protein kinase C increase the phosphorylation of the synapsins at sites phosphorylated by cAMP-dependent and Ca2+/calmodulin-dependent protein kinase in the rat hippocampal slice.

Previous studies have shown that activators of protein kinase C (C kinase) produce synaptic potentiation in the hippocampus. For example, the C kinase activator phorbol dibutyrate has been shown to increase transmitter release in the hippocampus. In addition, a role for C kinase in long-term potentiation has been proposed. A common assumption in such studies has been that substrates for C kinase were responsible for producing these forms of synaptic potentiation. However, we have recently shown that phorbol dibutyrate increased the phosphorylated of synapsin II (formerly protein III, Browning et al., 1987) in chromaffin cells (Haycock et al., 1988). Synapsin II is a synaptic vesicle-associated phosphoprotein that is a very poor substrate for C kinase but an excellent substrate for cAMP-dependent and Ca2+/calmodulin-dependent protein kinase. We felt, therefore, that activation of C kinase might lead to activation of a kinase cascade. Thus effects of C kinase activation might be produced via the phosphorylation of proteins that are not substrates for C kinase. In this report we test the hypothesis that activators of C kinase increase the phosphorylation of synapsin II and an homologous protein synapsin I. Our data indicate that PdBu produced dose-dependent increases in the phosphorylation of synapsin I and synapsin II. We also performed phospho-site analysis of synapsin I using limited proteolysis. These studies indicated that PdBu increased the phosphorylation of multiple sites on synapsin I. These sites have previously been shown to be phosphorylated by both cAMP-dependent protein kinase and the multifunctional Ca2+/calmodulin-dependent protein kinase II.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of electroconvulsive shock on the phosphorylation of DARPP-32 in rat striatum

Dopamine- and cAMP-regulated phosphoprotein with molecular weight 32 kDa (DARPP-32) is a key integrative molecule in the dopaminergic and glutamatergic signaling pathways in the striatum. Electroconvulsive shock (ECS), which induces massive neuronal depolarization, can activate various signaling pathways. In this study we investigated whether ECS could affect the phosphorylation status of DARPP-32. Male Sprague–Dawley rats underwent ECS and were sacrificed by decapitation at 0, 2, 10, 60, and 180 min after treatment. The phosphorylations of Thr34 and Thr75 residues of DARPP-32 and Ser159 residue of cyclin-dependent kinase 5 (CDK5) were investigated in the striatum. The activity of protein phosphatase 1 (PP1) and the binding between DARPP-32 and PP1 were also analyzed. Thr34 phosphorylation of DARPP-32 increased immediately after ECS and this state was maintained for more than 60 min. The activity of PP1 decreased and the binding between PP1 and DARPP-32 increased in accordance with this phosphorylation pattern. However, the phosphorylation at Thr75 showed no significant change except for an initial transient decrease. The phosphorylation of CDK5, which is responsible for Thr75 phosphorylation of DARPP-32, did not exhibit significant fluctuations. Our findings indicate that ECS increases Thr34 phosphorylation of DARPP-32, and thus inhibits the activity of PP1.     

Translocation of protein kinase C in rat hippocampal slices

Tumor-promoting phorbol esters specifically activate protein kinase C and mimic the effects of neurotransmitters in certain systems. Treatment of hippocampal slices with phorbol dibutyrate caused translocation of protein kinase C activity from cytoplasm to membranes. Experiments with carbachol, norepinephrine, glutamate, KCl, and LiCl failed to demonstrate a similar translocation. Translocation more readily provides an index of protein kinase C involvement for phorbol esters than for other agents in hippocampus.     

Tyrosine phosphorylation is increased in the rat hippocampus during the status epilepticus induced by pilocarpine

Phosphorylation of tyrosine residue in proteins is an important modulatory process for membrane transduction and cell signaling and for several cellular functions. The concentration and distribution of phosphotyrosine proteins were analyzed in the hippocampi of rats in the model of epilepsy induced by pilocarpine using Western blotting and immunohistochemistry. The concentration of several phosphotyrosine proteins increased during status epilepticus. During the seizure-free period and the chronic period of this epilepsy model, the hippocampi of rats did not exhibit changes in the expression of these proteins. Immunohistochemistry showed an increased immunoreactivity throughout the hippocampal formation of rats 1 h after status epilepticus that was acutely induced by pilocarpine. Animals killed after 3 h of status epilepticus showed an increased expression of phosphotyrosine in the hippocampal hilus and CA3 regions. After 5 h of status epilepticus, phosphotyrosine immunoreactivity persisted only in the CA3 region. After 12 h of status epilepticus, the hippocampal formation exhibited a normal phosphotyrosine immunostaining, showing that the increased expression of these proteins is related to the acute phase and that several intracellular events could undergo modifications during the status epilepticus induced by pilocarpine.     

Sympathetic sprouting reverses decreases in membrane-associated activity of protein kinase C following septohippocampal denervation of the rat hippocampus

Hippocampal sympathetic ingrowth (HSI), a form of neuronal plasticity, is induced by medial septal lesions and consists of the sprouting of peripheral sympathetic fibers, arising from the superior cervical ganglion, into the dentate gyrus and CA₃ region of the hippocampus. HSI has been previously shown to alter learned and spontaneous behaviors, phosphatidyl inositide hydrolysis, and the antagonist binding kinetics of both muscarinic cholinergic receptors and phorbol ester receptors. We now report that sympathetic sprouting reverses decreases in membrane-associated activity of protein kinase C (PKC) following septohippocampal denervation of the rat hippocampus. Further, no changes were found in α, β or γ PKC isoenzymes among experimental groups, suggesting that the group A PKC isoforms do not mediate the observed changes in activity and phorbol ester binding.