Secondary impairments after spinal cord injury: a population-based study.

OBJECTIVE: To determine the prevalence of secondary impairments among individuals with long-standing spinal cord injury in Quebec and the potential relationships between these impairments and several variables. DESIGN: A review of 2,200 medical files was conducted to determine the target population; 976 patients were selected randomly and mailed questionnaires. The results were based on 482 individuals with spinal cord injury who returned the completed questionnaire. The questionnaire included 14 subsections, such as sociodemographic, medical, psychosocial, and environmental information. The medical section, including the type and level of lesion and the presence of secondary impairments, was analyzed. RESULTS: Urinary tract infection, spasticity, and hypotension were the most frequently reported secondary impairments, regardless of the severity of injury. Relationships between the prevalence of secondary impairments and the duration of injury, as well as perceived health status, were observed. CONCLUSIONS: This is the first study to describe secondary impairments after long-standing spinal cord injury in Quebec. Patients with spinal cord injury still present a high prevalence of secondary impairments many years after their rehabilitation, despite preventive education or medical follow-up visits. Further studies are required to determine the specific impact that these impairments have on the patients' social role and their quality-of-life.

A role for PKC epsilon and MAP kinase in bradykinin-induced arachidonic acid release in rabbit CCD cells.

Arachidonic acid (AA) release is the rate-limiting step in the production of prostaglandins, an important class of autocrine/paracrine factors that modulate collecting duct function. Previous results from this laboratory have established cytosolic phospholipase A2 (cPLA2) as the enzyme responsible for bradykinin (BK)-stimulated AA mobilization in rabbit cortical collecting duct (RCCD) cells, and the present study pursues the intracellular signaling mechanisms responsible for its activation. Pretreatment of cells with Ro-31-8220, an inhibitor of protein kinase C (PKC), or PD-98059, an inhibitor of the mitogen-activated protein kinase (MAPK) cascade, resulted in a 50-60% reduction in BK-stimulated AA release. Incubation of RCCD cells with a combination of both Ro-31-8220 and PD-98059 did not achieve a greater inhibition of either BK-stimulated AA release or cPLA2 activity, possibly indicating that MAPK activation was dependent upon prior activation of PKC. This was supported by the observation that BK-induced MAPK activation could be reversed by either inhibitor. Additional experiments dealing with immunoblots for PKC isozymes revealed that RCCD cells express PKC species alpha, gamma, epsilon, and zeta. Following BK stimulation, only PKC epsilon translocated to the particulate fraction. Based on these results, it appears that PKC is activated and involved in the sequential activation of MAPK and cPLA2 following BK treatment. The results also suggest that PKC epsilon may be the isozyme implicated in the process.

Bradykinin-stimulated arachidonic acid release from MDCK cells is not protein kinase C dependent.

Bradykinin (BK)-induced release of arachidonic acid (AA) from Madin-Darby canine kidney (MDCK) D1 cells was investigated. Phorbol 12-myristate 13-acetate (PMA) caused a synergistic increase in BK- and A-23187-induced release of AA but alone had no effect on this release. Inhibition of protein kinase C (PKC) with bisindolmaleimide I (BIS) abolished the synergistic effects of PMA but did not affect AA release caused by BK or A-23187 alone. Downregulation of PKC with 100 nM PMA resulted in a reduction of AA release induced by BK or A-23187 addition, which corresponded to a decrease in cytoplasmic phospholipase A2 (cPLA2) activity as measured in cell extracts. Although Western blotting revealed no differences in cPLA2 expression as a result of PMA treatment, phosphorylation of the enzyme, as assessed by phosphoserine content, was significantly reduced in PKC-depleted cells. These results imply that, with PKC downregulation, subsequent BK stimulation results in a Ca(2+)-dependent translocation of a less phosphorylated, less active form of cPLA2. Any stimulation of PKC by BK addition did not appear as a significant event in onset responses leading to AA release. On the other hand, inhibition of the mitogen-activated protein kinase (MAPK) cascade with the MAPK kinase inhibitor, PD-98059, significantly decreased BK-induced release of AA, a finding that, with our other results, points to the existence of a PKC-independent route for stimulation of MAPK and the propagation of onset responses.

Bradykinin-induced translocation of cytoplasmic phospholipase A2 in MDCK cells.

The nonapeptide bradykinin (BK) plays an important role in the production of eicosanoids within the collecting duct of the nephron. We have shown previously that BK can initiate a complex signaling cascade that causes the release of arachidonic acid (AA) from MDCK-D1 cells, a canine cell line of distal tubule and collecting duct origin. This release is dependent upon early activation of specific upstream enzymes, including phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PLD). Ultimately, the release of this precursor of eicosanoids is effected by recruitment of the cytoplasmic 85-kDa form of phospholipase A2 (cPLA2). This enzyme is thought to translocate from the cytosol to cellular membranes following stimulation by agonists that cause elevations of intracellular calcium ([Ca2+]i). The present study was undertaken to examine the dependence of AA release upon Ca2+ influx in BK-stimulated MDCK cells. For this purpose, cells were incubated with 1 microM BK for 1 min and lysed in Ca(2+)-free Tris buffer. The high-speed 100000 x g pellet was extracted with 10 mM octyl glucoside and the cPLA2 protein level was determined. Previous results from our laboratory indicated that BK induced a 1.81-fold increase in cPLA2 activity associated with cellular membranes, while in the present study, Western blotting with a specific cPLA2 antibody demonstrated a similar elevation in protein detected with these same membranes. A selective inhibitor of receptor-mediated Ca2+ entry, SK&F 96365, was used to resolve the role of extracellular Ca2+ in BK's ability to evoke AA release. Pretreatment of cells with SK&F 96365 resulted in an inhibition of greater than 60% of the BK response. Taken together, these results strongly suggest that BK-mediated AA release in MDCK-D1 cells is at least partly contingent upon translocation of cPLA2 to membranes initiated by an influx of extracellular Ca2+.

Bestatin-mediated inhibition of leucine aminopeptidase may hinder HIV infection.

Bestatin, an inhibitor of leucine aminopeptidase (LAPase), significantly decreased HIV infection as reflected by a reduced number of positive immunofluorescent cells, p24 levels, reverse transcriptase activity and the number of proviral copies found in Bestatin-treated cells. Cellular and extracellular LAPase activity in infected cells was higher than the LAPase activity found in uninfected cells. However, cellular and extracellular LAPase activity as well as total protein kinase C activity was lower in Bestatin-treated cells. Conversely, the incubation of human lymphocytic HUT78 cells with LAPase promotes HIV infectivity. The possible role of LAPase in the pathophysiology of HIV was assessed by determining LAPase serum levels in HIV infected patients. LAPase activity levels were three orders of magnitude greater in sera obtained from HIV patients than those detected in sera of uninfected individuals. Although Bestatin reduced HIV infection, a moderate decrease in the reverse transcriptase activity of chronically-infected H9 human T-lymphocytic cells was observed. Based on the higher levels of LAPase present in the serum of HIV patients and on the combined inhibitory effect of Bestatin on LAPase and on protein kinase C activities, we suggest that LAPase may play an important role in the early events of HIV infection such as viral entry.

Bradykinin-stimulated cPLA2 phosphorylation is protein kinase C dependent in rabbit CCD cells.

We have used an established cell line of rabbit cortical collecting duct (RCCD) epithelial cells representing a mixed population of principal and intercalated cell types to determine which phospholipase A2 (PLA2) enzyme therein is responsible for bradykinin (BK)-stimulated arachidonic acid (AA) release and how its activation is regulated. BK-stimulated AA release was reduced 92% by arachidonyl trifluoromethyl ketone, an inhibitor of cytosolic PLA2 (cPLA2). Examination of PLA2 activity in vitro demonstrated that BK stimulation resulted in a greater than twofold increase in PLA2 activity and that this activity was dithiothreitol insensitive and was inhibited by an antibody directed against cPLA2. To determine a possible role for protein kinase C (PKC) in the BK-mediated activation of cPLA2, we used the PKC-specific inhibitor Ro31-8220 and examined its effects on AA release, cPLA2 activity, and phosphorylation. Ro31-8220 reduced BK-stimulated AA release and cPLA2 activity by 51 and 58%, respectively. cPLA2 activity stimulated by phorbol ester [phorbol 12-myristate 13-acetate (PMA)] displayed a similar degree of activation and was associated with an increase in serine phosphorylation identical to that caused by BK. The phosphorylation-induced activation of this enzyme was confirmed by the phosphatase-mediated reversal of both BK- and PMA-stimulated cPLA2 activity. In addition, we have also found that PMA stimulation did not cause a synergistic potentiation of BK-stimulated AA release as did calcium ionophore. This occurred despite membrane PKC activity increasing 93% in response to PMA vs. 42% in response to BK. These data, taken together, indicate that cPLA2 is the enzyme responsible for BK-mediated AA release, and, moreover, they indicate that PKC is involved in the onset responses of cPLA2 to BK.

Role of PLA2, PLC, and PLD in bradykinin-induced release of arachidonic acid in MDCK cells.

The role of cytosolic phospholipase A2 (cPLA2), phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PLD) in the bradykinin (BK)-stimulated release of arachidonic acid (AA) was examined in Madin-Darby canine kidney (MDCK) cells. Release of AA, phosphorylcholine, choline, and phosphatidic acid (PA) or the transphosphatidylation product, phosphatidylethanol, was detected after 1 min of BK stimulation. A role for PC-PLC was confirmed with D609, which reduced BK-stimulated AA by 70%. Ethanol (EtOH), which blunts PA formation, diminished BK-stimulated AA release by 50%. Together, D609 and EtOH inhibited this release almost completely. Evidence indicated that diacylglycerol and PA can enhance PLA2 activity when added to cytosol extracts. The enzyme responsible for AA release was characterized as cPLA2, since PLA2 activity assayed in cell extracts was largely inhibited by an antibody to this enzyme. The membrane fraction PLA2 activity increased significantly in BK-stimulated cells. We conclude that BK signaling in MDCK cells is mediated by the lipid products of PC-PLC and PLD, increasing cPLA2 activity, possibly by causing perturbations in the bilayer structure of its substrate, by a direct effect on the enzyme or by activation of protein kinases such as protein kinase C.

Regulation of bradykinin-stimulated phospholipase C and arachidonic acid release by protein kinase A in MDCK-D1 cells.

Regulation of phospholipases C (PLC) and arachidonic acid (AA) release by cAMP-dependent protein kinase (PKA) was investigated in MDCK-D1 cells. Bradykinin (BDK) was used to stimulate PLC and AA release, while arginine vasopressin (AVP), forskolin (FSK), isobutylmethylxanthine (IBMX) were used to increase cAMP levels and stimulate PKA. When cells were preincubated for 20 min with 10 microM FSK + 0.5 mM IBMX, and subsequently treated with 1 microM BDK or control medium for 40 min, the basal and BDK-stimulated PLC activity, measured as accumulated labelled inositol phosphate (InsP) after 40 min and inositol trisphosphate (InsP3) after 10 s, were significantly inhibited. In a parallel manner, FSK + IBMX also significantly decreased both basal and BDK-stimulated diacylglycerol (DAG) production. The basal and BDK-enhanced AA release into the media was also significantly inhibited by pretreatment with FSK + IBMX. In parallel experiments, H-89, a specific inhibitor of PKA, was preincubated for 60 min prior to addition of BDK and this resulted in a reversal of FSK+IBMX-induced inhibition of basal and BDK-stimulated PLC activity and AA release. An inhibitor of inositide-hydrolysing PLC, U73122, (1 microM) was also found to blunt BDK-stimulated PLC activity and BDK-enhanced AA release which indicated that stimulation of AA release by the nonapeptide was second to PLC activation. The ionophore, A23187, (10 microM) greatly stimulated AA release and to a much lesser extent, PLC activity. Its effect on AA release however was not blocked by inhibiting protein kinase C (PKC) with staurosporine (SSP) and consequently did not notably involve the PLC-PKC cascade. Activation of PKA with FSK + IBMX was found to significantly inhibit the enhancement of AA release by ionophore. With 12-tetradecanoyl-phorbol-13-acetate (TPA) also present there was a synergistic increase in the A23187-stimulated AA release and activation of PKA under such conditions inhibited AA release to a similar extent though the synergistic effect remained. The results strongly suggest a role for PKA in the regulation of PLC activity and AA release in MDCK-D1 cells. Control of AA release by PKA, is mediated both by mechanisms which involve blunting of PLC activity and mechanisms which are downstream from the PLC-PKC cascade.

Effect of retinoic acid on Nm/23 nucleoside diphosphate kinase and components of cyclic adenosine monophosphate-dependent signalling in human neuroblastoma cell lines.

The effects of retinoic acid on components of the cAMP-dependent signalling system were examined in two related human neuroblastoma cell lines SK-N-SH-F (SHF) and SK-N-SH-N (SHN). Retinoid treatment for a week significantly increased the concentration of intracellular cAMP and the levels of activity of protein kinase A and adenylate cyclase in both cell lines. Retinoic acid treatment also caused a very marked translocation of nucleoside diphosphate kinase from the cytosol to the membrane fraction. The increases in cyclic nucleotide and protein kinase A activity were observed to occur as early as within 1 and 2 days respectively and preceded or were concurrent with the onset of observable morphological differentiation. Results also indicated that agents which elevated intracellular cAMP caused neuronal differentiation and blunted retinoic acid-induced melanocytic differentiation in SHF cells. However, increases in cAMP brought about by treatment of SHF cells with retinoic acid alone were several-fold smaller and thus insufficient to induce neuritogenesis in these cells. The results as a whole indicate that one overall effect of retinoic acid treatment is to upgrade the activity of components of the cAMP-dependent signalling system in both neuroblastoma cell lines. However, retinoic acid causes the SH-F and SH-N cell lines to differentiate along different routes which means that the upgrading responses may be related to more general aspects of differentiation rather than to specific phenotype expression.

Vitamin E suppresses diacylglycerol (DAG) level in thrombin-stimulated endothelial cells through an increase of DAG kinase activity.

The present study has examined the role of vitamin E, a natural lipid antioxidant, in the production of diacylglycerol (DAG) and phosphatidic acid (PA) in thrombin-stimulated human endothelial cells. Cells were labelled with [3H]myristate and the incorporation and distribution of [3H]myristate into cellular lipids was not affected by vitamin E. However, in response to thrombin stimulation, considerably more PA and less DAG were formed in cells enriched with vitamin E. The time-course of thrombin stimulation indicated that vitamin E attenuated the accumulation of sustained DAG levels with a concomitant increase in PA. Direct determination of DAG mass further confirmed that vitamin E suppresses the accumulation of DAG induced by thrombin. In the presence of ethanol, the formation of [3H]phosphatidylethanol (PEt) in [3H]myristate-labelled cells stimulated by thrombin was unaffected by vitamin E enrichment. DL-Propranolol, a PA phosphohydrolase inhibitor, caused an accumulation of PA, without affecting DAG formation in either vitamin E-treated and untreated cells. This indicated that the increase in PA and decrease in DAG in vitamin E-treated cells was not due to a stimulation of phospholipase D or an inhibition of PA phosphohydrolase. Determination of inositol phosphates formation in response to thrombin showed that the change of DAG levels elicited by vitamin E was independent of phospholipase C-induced hydrolysis of inositol phospholipids. In contrast, analysis of DAG kinase activity revealed that vitamin E enrichment enhanced the activity of the enzyme in both basal and thrombin-stimulated cells. Taken together, these data indicated that vitamin E caused an increased conversion of DAG to PA by activating DAG kinase activity without causing any change in the activities of phospholipase D, PA phosphohydrolase or phospholipase C.

Transfer of arachidonyl groups within the lipids of two human neuroblastoma cell lines.

The incorporation and mobilization of [3H]arachidonic acid in lipids of human neuroblastoma cell lines, SK-N-SHF and LA-N-5, was studied. Essentially similar results were obtained with these two cell lines. Except for phosphatidylinositol which displayed the highest specific activity, the incorporation patterns within phospholipid classes tended to reflect phospholipid composition initially. However at later stages, counts in the acid-stable phosphatidylcholine plateaued and/or decreased while those of plasmenylethaniolamine and acid-stable phosphatidylethanolamine increased steadily. When cells were pulse-labelled with [3H]arachidonic acid and chased with fresh medium, there was a movement of label from diacyl (acid-stable) phosphatidylcholine to plasmenylethanolamine and diacyl (acid-stable) phosphatidylethanolamine. Plasmenylcholine did not appear to be involved in the arachidonyl group transfer. Under these chase conditions there was extensive turnover in the 32P-labelled polar headgroup of phosphatidylinositol but not in that of the other phospholipids. In both incorporation and chase studies involving [3H]arachidonic acid, a movement of arachidonyl groups from triacylglycerol to phospholipid could be observed. The results indicated that the patterns of incorporation and redistribution of arachidonic acid in human neuroblastoma cells were effectively regulated to favor lipids such as phosphatidylinositol and the subclasses of phosphatidylethanolamine. Possible mechanisms involved in these enrichment processes are discussed.

Retinoic acid- and staurosporine-induced bidirectional differentiation of human neuroblastoma cell lines.

The differentiation pattern of two related human neuroblastoma cell lines, SK-N-SHF and SK-N-SHN, induced by retinoic acid and staurosporine was studied. Immunohistochemical and electron microscopic examination of the cells indicated that the SHF variant could undergo differentiation along a melanocytic route when treated with retinoic acid and to neuronal cells when treated with retionic acid and staurosporine together. Treatment of SHN cells with either or both these agents caused neuronal differentiation. The melanocytic pathway was characterized in part by the flattening of the cells, the appearance of melanocytic antigens and various forms of melanosomes, an increase in tyrosinase activity, and the absence of neuronal marker proteins. The neuronal route was typified by the development of long neuritic processes containing microtubules and numerous neurosecretory granules as well as by immunohistochemical reactions for neural cell adhesion molecule, synaptophysin, and neurofilament proteins. The significance of these results is discussed in terms of the differentiation responses of neuroblastoma cells to chemical agents as well as some of the factors involved in the regulation of phenotype expressions of these cells.

Further characterization of the diacylglycerol-phosphatidylethanolamine exchange reaction catalyzed by cell-free extracts of Escherichia coli.

The conditions for phosphatidylethanolamine (PE)-diacylglycerol (DAG) exchange catalysed by cell-free extracts of Escherichia coli were studied using 14C- or 3H-analogues of both these lipids. The reaction, examined with either labelled PE or labelled DAG, occurred without co-factor addition and was inhibited by Ca2+ and Mg2+. Detergents such as Triton X-100 greatly enhanced the activity; however, the optimal concentration of this agent depended on the lipid substrate concentration. The exchange-catalysing enzyme involved in these extracts appeared to be very specific for DAG and PE, since no other labelled phospholipid or acylglycerol derivative formed radioactive product under the assay conditions tested. Again, endogenous [3H]PE present in the enzyme source, but no other endogenous lipid, was converted to labelled DAG in the presence of added 1,2-dioleoyl-sn-glycerol. The Vmax value for the conversion of labelled PE to DAG was very similar to the Vmax value found for the conversion of labelled DAG to PE as would be expected in the case of an exchange reaction being responsible for both conversions. However, the Km value for PE was appreciably larger than that for DAG. The enzyme involved, displayed a broad acyl chain specificity as could be judged from: (1) the ability of various species of DAG and PE to stimulate the exchange; (2) the suitability of lipid substrates prepared from widely different biological sources; and (3) the interchange of acyl groups that occurred between dimyristoyl PE and dilauroylglycerol. As would be expected for an exchange reaction, the incorporation of lauroyl groups into PE occurred without an increase in the total fatty acid content of this phospholipid. The results of the present study confirm and further characterize the PE-DAG exchange reaction of E. coli.

Effects of retinoic acid and staurosporine on the protein kinase C activity and the morphology of two related human neuroblastoma cell lines.

Studies on the involvement of protein kinase C in retinoic acid-induced differentiation of human neuroblastoma were carried out with two variants of the SK-N-SH cell line namely the SH-F subline, which differentiates to give a fibroblast-like phenotype, and the SH-N subline, which develops into the typical neuronal phenotype. In SH-F, a substantial increase in protein kinase C activity accompanied morphological differentiation. Accordingly, after 7 days of retinoic acid treatment, EDTA-extracted, cytosolic protein kinase C activity increased by slightly more than 2-fold over vehicle-treated controls. Again, detergent-extracted activity, representing membrane-bound or total protein kinase C, showed a similar 2.6- to 5.1-fold increase in treated cells. A time-course study revealed an earliest increase in total activity after two days of retinoic acid treatment which continued linearly for the first 6 to 8 days, and then levelled off. A study of the effect of retinoic acid on the protein kinase C in vitro with SH-F cell extracts showed only a slight increase in activity (of 25%) at the relatively high concentration of 10(-4) M; however, no significant differences were observed at lower concentrations. In contrast, the SH-N cell line responded to retinoic acid by a 45% decrease in EDTA-extractable, and a 63% decrease in detergent-extractable protein kinase C activity. Added to SH-F cell cultures, 15 nM staurosporine was found to inhibit protein kinase C in vivo and to a lesser extent, the protein kinase A. Present together with retinoic acid, staurosporine not only prevented the augmentation but caused a marked decrease of protein kinase C activity in this cell line. Morphological studies indicated that when SH-N cells are treated with staurosporine, or staurosporine and retinoic acid together, a neuronal phenotype similar to that produced by retinoic acid alone is observed. In contrast, when the SH-F cell line is treated with staurosporine or staurosporine and retinoic acid together, the flattened fibroblast-like cell type normally induced by retinoic acids is not observed. Instead, these cells display much smaller cell bodies and elaborate extensions resembling the neuronal phenotype produced by retinoic acid induced differentiation of the SH-N variant. These results suggest that changes in the protein kinase C activity may be involved in regulating the expression of the phenotype during cell differentiation.

Diacylglycerol-phosphatidylethanolamine exchange in Escherichia coli.

The conversion of [3H]- or [14C]diacylglycerol to labelled phosphatidylethanolamine by dialysed, particulate fraction of Escherichia coli was studied. The reaction occurred in the presence of hydroxylamine, under which conditions, the synthesis of [14C]phosphatidylethanolamine from CDP-diacylglycerol and [14C]serine did not occur. The conversion was enhanced by addition of dilauroyl- or dioleoylphosphatidylethanolamine. A conversion of [3H]- or [14C]phosphatidylethanolamine to labelled diacylglycerol could also be readily demonstrated provided unlabelled diacylglycerol was added. Double-labelled [acyl-3H, 32P]phosphatidylethanolamine was converted to labelled diacylglycerol without formation of labelled water-soluble products. The formation of double-labelled phosphatidylethanolamine from [3H]diacyl[14C]glycerol or of double-labelled diacylglycerol from [acyl-3H,glycerol-14C]phosphatidylethanolamine occurred without significant change in isotope ratio. When [acyl-3H]phosphatidylethanolamine was incubated with increasing concentrations of [acyl-14C]diacylglycerol, correspondingly increasing concentrations of [14C]phosphatidylethanolamine were formed which matched the concentrations of [3H]diacylglycerol produced concurrently. It was concluded that E. coli extracts can catalyze an exchange between the diacylglycerol moiety of phosphatidylethanolamine and free diacylglycerol with complete sparing of the phosphoethanolamine moiety.

Modulation by phorbol 12-myristate 13-acetate of arachidonic acid release from rat basophilic leukemia cells stimulated with A23187.

Rat basophilic leukemia (RBL-2H3) cells were cultured in medium containing [3H]arachidonic acid and labelling of the different lipid fractions was followed with time. After up to 4 h of culture, the label was found mostly in phosphatidylcholine. After 8 h, labelling of phosphatidylethanolamine gradually exceeded that of phosphatidylcholine, until at 24 h, approximate equilibrium labelling of the lipid fractions was attained and 45% of the label was found in phosphatidylethanolamine, 35% in phosphatidylcholine, 18% in the phosphatidylserine/inositide fraction and the remainder in the neutral lipid fraction. Stimulation of cells with A23187 after 30 min of labelling caused release of [3H]arachidonic acid which was accountable by a decrease in radioactivity of phosphatidylcholine, whereas stimulation of cells after 24 h of labelling caused the release of radioactive arachidonic acid, which was accompanied by a decrease of label in both phosphatidylcholine and phosphatidylethanolamine. Incubation of the labelled cells with phorbol 12-myristate 13-acetate prior to ionophore addition enhanced both the release of [3H]arachidonic acid and its metabolites and the decrease in label of the same phospholipids as those affected by ionophore alone. Under our conditions, the enhancement effects of phorbol ester were greatest after 2-5 min of preincubation, prior to ionophore addition. The results suggest that in basophilic leukemia cells, arachidonic acid release proceeds from several pools of phospholipids and that the activity of the phospholipase(s) involved is modulated by protein kinase C.