Expression and distribution of protein kinase C isozymes in brain tissue of spontaneous hypertensive rats.

Hypertension activates many endocrine, neuroendocrine and metabolic responses. How hypertension alters these functions remains unknown. Consequently the pathophysiology of hypertension related illnesses are incompletely understood. Protein kinase C (PKC) isoforms play an important role in cellular signal transduction and altered PKC activity has been reported in spontaneous hypertensive rats (SHR). In order to understand the role that PKC plays in hypertension, we hypothesized that PKC activity is significantly expressed in synaptosomal preparations from the brains of SHRs. In addition, the neuroanatomical distribution of this expression was mapped and compared to control animals. The brains were further studied for signs of neuropathology. Total PKC activity was significantly increased in synaptosomal samples isolated from the forebrain, midbrain, and hindbrain of SHR rats. Westem blot analysis identified PKC-alpha, -beta, -gamma, -delta, -epsilon and -zeta in all brain regions. Immunohistochemical analyses indicated that PKC-gamma was localized in cell bodies and processes in many autonomic cardiovascular control regions. These results suggest that PKC may be an important modulator of autonomic blood pressure control.     

Activation of PKC modulates blood-brain barrier endothelial cell permeability changes induced by hypoxia and posthypoxic reoxygenation

The blood-brain barrier (BBB) is a metabolic and physiological barrier important for maintaining brain homeostasis. The aim of this study was to determine the role of PKC activation in BBB paracellular permeability changes induced by hypoxia and posthypoxic reoxygenation using in vitro and in vivo BBB models. In rat brain microvessel endothelial cells (RMECs) exposed to hypoxia (1% O2-99% N2; 24 h), a significant increase in total PKC activity was observed, and this was reduced by posthypoxic reoxygenation (95% room air-5% CO2) for 2 h. The expression of PKC-betaII, PKC-gamma, PKC-eta, PKC-mu, and PKC-lambda also increased following hypoxia (1% O2-99% N2; 24 h), and these protein levels remained elevated following posthypoxic reoxygenation (95% room air-5% CO2; 2 h). Increases in the expression of PKC-epsilon and PKC-zeta were also observed following posthypoxic reoxygenation (95% room air-5% CO2; 2 h). Moreover, inhibition of PKC with chelerythrine chloride (10 microM) attenuated the hypoxia-induced increases in [14C]sucrose permeability. Similar to what was observed in RMECs, total PKC activity was also stimulated in cerebral microvessels isolated from rats exposed to hypoxia (6% O2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min). In contrast, hypoxia (6% O2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min) significantly increased the expression levels of only PKC-gamma and PKC-theta in the in vivo hypoxia model. These data demonstrate that hypoxia-induced BBB paracellular permeability changes occur via a PKC-dependent mechanism, possibly by differentially regulating the protein expression of the 11 PKC isozymes.     

Effects of the PKC inhibitor PD 406976 on cell cycle progression, proliferation, PKC isozymes and apoptosis in glioma and SVG-transformed glial cells

It is well established that protein kinase C (PKC) isozymes are involved in the proliferation of glioma cells. However, reports differ on which PKC isozymes are responsible for glioma proliferation. As a means to further elucidate this, the objectives of our research were to determine how inhibition of PKC-alpha, PKC-beta and PKCmu with PD 406976 regulates the cell cycle, cell proliferation and PKC during glioma growth and development. To establish the cell cycle effects of PD 406976 on brain cells (SVG, U-138MG and U-373MG glioma cells), specimens were treated with either dimethylsulfoxide (DMSO; control) or PD 406976 (2 microm). Results from flow cytometry demonstrated that PD 406976 delayed the entry DNA synthesis phase in SVG cells and delayed the number of cells entering and exiting the DNA synthesis phase in both U-138MG and U-373MG cells, indicating that PD 406976 may inhibit G(1)/S and S phase progression. Assessment of cell viability demonstrated a cytostatic effect of PD 406976 on SVG, U-138MG and U-373MG glioma cell proliferation. The PD 406976-induced decreased proliferation was sustained at 48-96 h. A PKC activity assay was quantified and demonstrated that exposure of SVG and U-373MG glioma cells to PD 406976 suppressed PKC activity. Western blotting demonstrated reduced PKC-beta1, PKC-gamma and PKC-tau protein content in cells treated with PD 406976. We determined that the growth inhibitory effect of PD 406976 was not as a result of apoptosis.     

The linoleic acid derivative DCP-LA selectively activates PKC-epsilon, possibly binding to the phosphatidylserine binding site

This study examined the effect of 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), a newly synthesized linoleic acid derivative with cyclopropane rings instead of cis-double bonds, on protein kinase C (PKC) activity. In the in situ PKC assay with reverse-phase high-performance liquid chromatography, DCP-LA significantly activated PKC in PC-12 cells in a concentration-dependent (10 nM-100 microM) manner, with the maximal effect at 100 nM, and the DCP-LA effect was blocked by GF109203X, a PKC inhibitor, or a selective inhibitor peptide of the novel PKC isozyme PKC-epsilon. Furthermore, DCP-LA activated PKC in HEK-293 cells that was inhibited by the small, interfering RNA against PKC-epsilon. In the cell-free PKC assay, of the nine isozymes examined here, DCP-LA most strongly activated PKC-epsilon, with >7-fold potency over other PKC isozymes, in the absence of dioleoyl-phosphatidylserine and 1,2-dioleoyl-sn-glycerol; instead, the DCP-LA action was inhibited by dioleoyl-phosphatidylserine. DCP-LA also activated PKC-gamma, a conventional PKC, but to a much lesser extent compared with that for PKC-epsilon, by a mechanism distinct from PKC-epsilon activation. Thus, DCP-LA serves as a selective activator of PKC-epsilon, possibly by binding to the phosphatidylserine binding site on PKC-epsilon. These results may provide fresh insight into lipid signaling in PKC activation.     

Protein kinase C-gamma is present in adriamycin resistant HL-60 leukemia cells

The isoform pattern of protein kinase C (PKC) was examined in wild-type and Adriamycin-resistant (HL-60/AR) HL-60 leukemia cells. Analyses were carried out by immunoblotting with mouse monoclonal antibodies against PKC-alpha and PKC-beta and a rabbit polyclonal antibody against the variable (V3) region of PKC-gamma. HL-60/AR cells contained an equivalent level of PKC-alpha and a lower amount of PKC-beta than HL-60 cells. In contrast, only HL-60/AR cells contained PKC-gamma. These results indicate that the regulation of this family of isoenzymes is altered in drug-resistant cells.     

Activation of protein kinase C isozymes by contractile stimuli in arterial smooth muscle.

Protein kinase C (PKC) has been proposed to be involved in the regulation of vascular smooth muscle (VSM) contractile activity. However, little is known in detail about the activation of this kinase or specific isozymes of this kinase by contractile stimuli in VSM. As an index of PKC activation, Ca(2+)- and phospholipid-dependent histone IIIS kinase activity was measured in the particulate fraction from individual strips of isometrically contracting carotid arterial smooth muscle. Phorbol 12,13-dibutyrate (PDB) increased PKC activity in the particulate fraction (155% over resting value by 15 min) with a time course which paralleled or preceded force development. Stimulation with the agonist histamine (10(-5) M) resulted in rapid increases in both force and particulate fraction PKC activity which was maximal by 2 min (increase of 139%) and partially sustained over 45 min (increase of 41%). KCl (109 mM), which evokes a sustained contractile response, caused a slow increase (124% by 45 min) in particulate fraction PKC activity. No significant increases in activator-independent histone kinase activity were observed in response to any stimulus tested. PKC alpha and PKC beta were identified as the principal Ca2+/phospholipid-dependent PKC isozymes expressed in this tissue. In unstimulated arterial tissue, the ratio of immunodetectable isozyme content (alpha:beta) was estimated to be 1:1 in the particulate and 1.5:1 in the cytosolic fractions. Upon stimulation with each of the three contractile stimuli, particulate fraction PKC content assessed by immunoblotting increased with a time course and to an extent comparable to the observed changes in PKC activity. There was no evidence of differential regulation of the PKC alpha or -beta isozymes by PDB compared to the other contractile stimuli. These results indicate that diverse contractile stimuli are capable of tonically activating PKC in preparations of functional smooth muscle, and are consistent with a functional role for PKC alpha and/or -beta in the regulation of normal smooth muscle contractile activity.     

Gender-related distinctions in protein kinase C activity in rat vascular smooth muscle

Gender differences in vascular reactivity have been suggested; however, the cellular mechanisms involved are unclear. We tested the hypothesis that the gender differences in vascular reactivity reflect gender-related, possibly estrogen-mediated, distinctions in the expression and activity of specific protein kinase C (PKC) isoforms in vascular smooth muscle. Aortic strips were isolated from intact and gonadectomized male and female Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Isometric contraction was measured in endothelium-denuded aortic strips. PKC activity was measured in the cytosolic and particulate fractions, and the amount of PKC was measured using Western blots and isoform-specific anti-PKC antibodies. In intact male WKY rats, phenylephrine (Phe, 10(-5) M) and phorbol 12,13-dibutyrate (PDBu, 10(-6) M) stimulated contraction to 0.37 +/- 0.02 and 0.42 +/- 0.02 g/mg tissue wt, respectively. The basal particulate/cytosolic PKC activity ratio was 0.86 +/- 0.06, and Western blots revealed alpha-, delta-, and zeta-PKC isoforms. Phe and PDBu increased PKC activity and caused significant translocation of alpha- and delta-PKC from the cytosolic to particulate fraction. In intact female WKY rats, basal PKC activity, the amount of alpha-, delta-, and zeta-PKC, the Phe- and PDBu-induced contraction, and PKC activity and translocation of alpha- and delta-PKC were significantly reduced compared with intact male WKY rats. The basal PKC activity, the amount of alpha-, delta-, and zeta-PKC, the Phe and PDBu contraction, and PKC activity and alpha- and delta-PKC translocation were greater in SHR than WKY rats. The reduction in Phe and PDBu contraction and PKC activity in intact females compared with intact males was greater in SHR ( approximately 30%) than WKY rats ( approximately 20%). Phe and PDBu contraction and PKC activity were not significantly different between castrated males and intact males but were greater in ovariectomized (OVX) females than intact females. Treatment of OVX females or castrated males with 17 beta-estradiol, but not 17 alpha-estradiol, subcutaneous implants caused significant reduction in Phe and PDBu contraction and PKC activity that was greater in SHR than WKY rats. Phe and PDBu contraction and PKC activity in OVX females or castrated males treated with 17 beta-estradiol plus the estrogen receptor antagonist ICI-182,780 were not significantly different from untreated OVX females or castrated males. Thus a gender-related reduction in vascular smooth muscle contraction in female WKY rats with intact gonads compared with males is associated with reduction in the expression and activity of vascular alpha-, delta-, and zeta-PKC. The gender differences in vascular smooth muscle contraction and PKC activity are augmented in the SHR and are possibly mediated by estrogen.     

Dopaminergic inhibition of secretin-stimulated choleresis by increased PKC-gamma expression and decrease of PKA activity

To determine the role and mechanisms of action by which dopaminergic innervation modulates ductal secretion in bile duct-ligated rats, we determined the expression of D1, D2, and D3 dopaminergic receptors in cholangiocytes. We evaluated whether D1, D2 (quinelorane), or D3 dopaminergic receptor agonists influence basal and secretin-stimulated choleresis and lumen expansion in intrahepatic bile duct units (IBDU) and cAMP levels in cholangiocytes in the absence or presence of BAPTA-AM, chelerythrine, 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine (H7), or rottlerin. We evaluated whether 1) quinelorane effects on ductal secretion were associated with increased expression of Ca(2+)-dependent PKC isoforms and 2) increased expression of PKC causes inhibition of PKA activity. Quinelorane inhibited secretin-stimulated choleresis in vivo and IBDU lumen space, cAMP levels, and PKA activity in cholangiocytes. The inhibitory effects of quinelorane on secretin-stimulated ductal secretion and PKA activity were blocked by BAPTA-AM, chelerythrine, and H7. Quinelorane effects on ductal secretion were associated with activation of the Ca(2+)-dependent PKC-gamma but not other PKC isoforms. The dopaminergic nervous system counterregulates secretin-stimulated ductal secretion in experimental cholestasis.     

α-MELANOCYTE-STIMULATING HORMONE: A POSSIBLE NEURONAL MARKER OF THE AGEING BRAIN

Abstract— The amount of α-melanocyte-stimulating hormone (α-MSH) in the entire hypothalamus as well as the amount of α-MSH in free granule and synaptosome fractions of hypothalamic homogenates was investigated throughout the lifespan of female rats (1-24 months). A 900 g supernatant fluid was prepared from hypothalami following homogenization in an iso-osmotic sucrose solution, and free granules and synaptosomes containing α-MSH were fractionated by means of continuous sucrose density gradient centrifugation. α-MSH was quantified by radioimmunoassay. The total amount of α-MSH in the hypothalamus, as well as the amount in free granules and synaptosomes prepared from hypothalami increased progressively from the 1st to the 5th month of life, and this increase was more pronounced in the free granules than in the synaptosomes. On the other hand, the amount of α-MSH in the hypothalamus and the amount present in free granules and synaptosomes prepared from 5-24-month-old animals decreased with age, and this decrease appeared to proceed at similar rates in both subcellular compartments. Based on these results, it is suggested that ageing of α-MSH neurons in the hypothalamus is accompanied by a degeneration of the axons and/or an alteration in the biosynthetic and degradative activities of the neuron.     

Roles of protein kinase C in regulation of P2X7 receptor-mediated calcium signalling of cultured type-2 astrocyte cell line, RBA-2

The role of protein kinase C (PKC) on regulation of P2X(7) receptor-mediated Ca(2+) signalling was examined on RBA-2 astrocytes. Activation of PKC decreased the receptor-mediated Ca(2+) signalling and the decrease was restored by PKC inhibitors. Down regulation of PKC also caused a decrease in the Ca(2+) signalling. Thus PKC might play a dual role on the P2X(7) receptor signalling. Successive stimulation of the P2X(7) receptor induced a gradual decline of Ca(2+) signalling but PKC inhibitors failed to restore the decline. Nevertheless, PMA stimulated translocation of PKC-alpha, -betaI, -betaII, and -gamma, but only anti-PKC-gamma co-immunoprecipitated the receptors. To examine the role of PKC-gamma, Ca(2+) signalling was measured by Ca(2+) imaging. Our results revealed that the agonist-stimulated Ca(2+) signalling were reduced in the cells that the transfection of either P2X(7) receptor or PKC-gamma morpholino antisense oligo was identified. Thus, we concluded that PKC-gamma interacted with P2X(7) receptor complex and positively regulated the receptor-mediated Ca(2+) signalling.     

Increased expression of protein kinase C isoforms in heart failure due to myocardial infarction

The activities of cardiac protein kinase C (PKC) were examined in hemodynamically assessed rats subsequent to myocardial infarction (MI). Both Ca(2+)-dependent and Ca(2+)-independent PKC activities increased significantly in left ventricular (LV) and right ventricular (RV) homogenates at 1, 2, 4, and 8 wk after MI was induced. PKC activities were also increased in both LV and RV cytosolic and particulate fractions from 8-wk infarcted rats. The relative protein contents of PKC-alpha, -beta, -epsilon, and -zeta isozymes were significantly increased in LV homogenate, cytosolic (except PKC-alpha), and particulate fractions from the failing rats. On the other hand, the protein contents of PKC-alpha, -beta, and -epsilon isozymes, unlike the PKC-zeta isozyme, were increased in RV homogenate and cytosolic fractions, whereas the RV particulate fraction showed an increase in the PKC-alpha isozyme only. These changes in the LV and RV PKC activities and protein contents in the 8-wk infarcted animals were partially corrected by treatment with the angiotensin-converting enzyme inhibitor imidapril. No changes in protein kinase A activity and its protein content were seen in the 8-wk infarcted hearts. The results suggest that the increased PKC activity in cardiac dysfunction due to MI may be associated with an increase in the expression of PKC-alpha, -beta, and -epsilon isozymes, and the improvement of heart function in the infarcted animals by imidapril may be due to partial prevention of changes in PKC activity and isozyme contents.     

Adhesion of epithelial cells to fibronectin or collagen I induces alterations in gene expression via a protein kinase C-dependent mechanism

Adhesion of human salivary gland (HSG) epithelial cells to fibronectin- or collagen I gel-coated substrates, mediated by beta1 integrins, has been shown to upregulate the expression of more than 30 genes within 3-6 h. Adhesion of HSG cells to fibronectin or collagen I for 6 h also enhanced total protein kinase C (PKC) activity by 1.8-2.3-fold. HSG cells expressed PKC-alpha, gamma, delta, epsilon, mu, and zeta. Adhesion of HSG cells to fibronectin or collagen I specifically activated PKC-gamma and PKC-delta. Cytoplasmic PKC-gamma and PKC-delta became membrane-associated, and immunoprecipitated PKC-gamma and PKC-delta kinase activities were enhanced 2.5-4.0-fold in HSG cells adherent to fibronectin or collagen I. In addition, adhesion of fibronectin-coated beads to HSG monolayers co-aggregated beta1 integrin and PKC-gamma and PKC-delta but not other PKC isoforms. Thus, integrin-dependent adhesion of HSG cells to fibronectin or collagen I activated PKC-gamma and PKC-delta. The role of this PKC upregulation on adhesion-responsive gene expression was then tested. HSG cells were treated with the specific PKC inhibitor bisindolylmaleimide I, cultured on non-precoated, fibronectin- or collagen I-coated substrates, and analyzed for changes in adhesion-responsive gene expression. Bisindolylmaleimide I strongly inhibited the expression of seven adhesion-responsive genes including calnexin, decorin, S-adenosylmethionine decarboxylase, steroid sulfatase, and 3 mitochondrial genes. However, the expression of two adhesion-responsive genes was not affected by bisindolylmaleimide I. Treatment with bisindolylmaleimide I did not affect cell spreading and did not significantly affect the actin cytoskeleton. These data suggest that adhesion of HSG cells to fibronectin or collagen I induces PKC activity and that this induction contributes to the upregulation of a variety of adhesion-responsive genes.     

Free calcium concentrations in the nerve endings in the brain of rats with spontaneous hypertension

Fluorescent indicator Quin-2 was used for the determination of free calcium (Ca2+in) in synaptosomes incubated in the normal medium and media where sodium is replaced by potassium or choline. At external calcium concentration of 1 mM, Ca2+in in all three media was 20-30% higher in synaptosomes of spontaneously hypertensive rats (SHR) than in control animals. At external calcium concentration of 5 mM, the increase in Ca2+in values induced by K+-depolarization in sodium- or choline-containing media was 50-80% higher in synaptosomes of SHR. These differences are suggested to be the basis for the mechanism of increased peripheral chain activity in the sympathetic nervous system in primary hypertension.     

Effects of angiotensin II type 1 receptor blockade on the oxidative stress in spontaneously hypertensive rat tissues

The aim of this work was to investigate the production of oxidative damage in homogenized kidney, liver and brain of spontaneously hypertensive rats (SHR), as well as the involvement of angiotensin (Ang) II in this process. Groups of 12-week-old SHR and Wistar Kyoto rats (WKY) were given 10 mg/kg/day losartan in the drinking water during 14 days. Other groups of WKY and SHR without treatment were used as controls. The production of thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH) and the activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (Gpx) were determined. No significant difference in TBARS was observed between untreated SHR or WKY rats; GSH content was lower in the liver but higher in the brain of SHR compared to WKY rats. In tissues from the SHR group, SOD and Gpx activities were reduced, whereas CAT activity was slightly increased in kidney. TBARS levels did not change in WKY rats after losartan administration, but were reduced in SHR liver and brain. Losartan treatment decreased GSH content in WKY kidney, but increased GSH in SHR liver. The activity of the antioxidant enzymes was not modified by losartan in WKY rats; however, their activities increased in tissues from treated SHR. The lower activity of antioxidant enzymes in tissues from hypertensive rats compared to those detected in normotensive controls, indicates oxidative stress production. Ang II seems to play no role in this process in normotensive animals, although AT1 receptor blockade in SHR enhances the enzymatic activity indicating that Ang II is implicated in oxidative stress generation in the hypertensive animals.     

Differential stimulation of protein kinase C activity by phorbol ester or calcium/phosphatidylserine in vitro and in intact synaptosomes.

Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) was compared with calcium/phosphatidylserine (Ca/PS). The substrate specificity of PKC was more limited with PS/PMA. Substrates could be divided into three overlapping groups according to their relative level of phosphorylation: C1, relatively preferred substrates with Ca/PS, included dephosphin, histone, and peptide GS1-10. C2, relatively preferred with PS/PMA, included myelin basic protein and MARCKS. C3, substrates independent of activators. PS/PMA altered the Vmax of PKC for substrate, and decreased the Km for Mg2+. Differential substrate phosphorylation by PS/PMA also occurred for PKC isozymes resolved by hydroxylapatite chromatography and was most dramatic for PKC-alpha, which could no longer phosphorylate histone or GS1-12. Differential activities of PKC were also observed in synaptosol and in intact synaptosomes where PMA stimulated phosphorylation of MARCKS, but not dephosphin. It was further shown that dephosphin was indeed a substrate of PKC in the intact synaptosomes by use of a repolarization-dependent dephosphin phosphorylation assay. The differential PKC activities could also be distinguished by inhibitors. H-7 was equipotent, palmitoylcarnitine did not inhibit in vitro C2 phosphorylation, but inhibited dephosphin in intact synaptosomes, and sphingosine did not inhibit C1 substrates and was without effect on dephosphin in intact synaptosomes. Therefore PS/PMA alters or limits the substrate specificity of PKC, leading to a differential substrate phosphorylation in vitro and in intact synaptosomes and differential inhibitor sensitivity. The pattern of protein phosphorylation observed after PKC activation in intact cells will therefore be dependent upon the activator.     

Different Modulation of Protein Kinase C Isozymes in Dibutyryl Cyclic AMP-Differentiated PC12h Cells

Abstract: PC12h cells can be differentiated into sympathetic neuron-like cells by various agents, including nerve growth factor, basic fibroblast growth factor, cyclic AMP analogues, and protein kinase C (PKC) activators. To study the involvement of PKC in the process of PC12h cell differentiation by cyclic AMP treatment, PKC isozymes (α, βI, βII, and γ) were analyzed using column chromatography and immunoblotting. Two PKC isozymes, PKC(α) and PKC(βII), were predominantly detected in PC12h cells. When stimulated by dibutyryl cyclic AMP, PKC(α) levels declined in the cytosolic fraction of the cells, whereas PKC(βII) levels increased. Increased PKC(βII) levels were also detected in the particulate fraction, whereas particulate PKC(α) levels did not change. The total PKC activity decreased in the cytosolic fraction following cyclic AMP stimulation of PC12h cells, whereas it stayed constant in the particulate fraction. Fractionation on a hydroxyapatite column showed a decreased level of PKC(α) activity and a transient increase followed by a decreased level of PKC(βII) activity. This discrepancy between increased PKC(βII) immunoreactivity and reduced PKC(βII) activity suggested the presence of nonactivatable PKC(βII) in cyclic AMP-treated PC12h extract. These findings indicate that PKC(α) and PKC(βII) are differentially regulated during the differentiation of PC12h cells. In addition, the differentiation of PC12h cells triggered by cyclic AMP seems to involve characteristic alterations of PKC isozymes.     

Antioxidant activity of propionyl-L-carnitine in liver and heart of spontaneously hypertensive rats

Oxidative stress plays an important role in arterial hypertension and propionyl-L-carnitine (PLC) has been found to protect cells from toxic reactive oxygen species. In this work, we have evaluated the antioxidant capacity of chronic PLC treatment in spontaneously hypertensive rats (SHR) by measuring the activity of antioxidant enzymes and the lipid peroxidation in liver and cardiac tissues. The activity of glutathione peroxidase was decreased in liver and cardiac tissues of SHR when compared with their normotensive controls, Wistar- Kyoto (WKY) rats, this alteration being prevented by PLC treatment. Glutathione reductase activity was increased in hypertensive rats and no effect was observed after the treatment. No significant changes in superoxide dismutase activity were observed among all experimental groups. Liver of hypertensive rats showed higher catalase activity than that of normotensive rats, and PLC enhanced this activity in both rat strains. Thiobarbituric acid reactive substances, determined as a measure of lipid peroxidation, were increased in SHR compared with WKY rats, and PLC treatment decreased these values not only in hypertensive rats but also in normotensive ones. The content of carnitine in serum, liver and heart was higher in PLC-treated rats, but PLC did not prevent the hypertension development in young SHR. In addition, triglyceride levels, which were lower in SHR than WKY rats, were reduced by chronic PLC treatment in both rat strains. These results demonstrate: i) the hypotriglyceridemic effect of PLC and ii) the antioxidant capacity of PLC in SHR and its beneficial use protecting tissues from hypertension-accompanying oxidative damage.     

Relative contribution of Rho kinase and protein kinase C to myogenic tone in rat cerebral arteries in hypertension

Arterial smooth muscle constriction in response to pressure, i.e., myogenic tone, may involve calcium-dependent and calcium-sensitization mechanisms. Calcium sensitization in vascular smooth muscle is regulated by kinases such as PKC and Rho kinase, and activity of these kinases is known to be altered in cardiovascular disorders. In the present study, we evaluated the relative contribution of PKC and Rho kinase to myogenic tone in cerebral arteries in hypertension. Myogenic tone and arterial wall calcium in Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were measured simultaneously, and the effect of PKC and Rho kinase inhibitors on myogenic tone was evaluated. SHR arteries showed significantly greater myogenic tone than WKY arteries. Pressure/wall tension-arterial wall calcium curves showed a hyperbolic relation in WKY rats, but the curves for SHR arteries were parabolic. Myogenic tone was decreased by the Rho kinase inhibitors Y-27632 and HA-1077, with a significantly greater effect in SHR than in WKY arteries. Reduction in myogenic tone produced by the PKC inhibitor bisindolylmaleimide I in WKY and SHR arteries was significantly less than that produced by Rho kinase inhibition. The pressure-dependent increase in myogenic tone was significantly decreased by Y-27632, and the decrease was markedly greater than that produced by bisindolylmaleimide I in SHR arteries. In WKY arteries, the pressure-dependent increase in myogenic tone was decreased to a similar extent by Y-27632 and bisindolylmaleimide I. These results suggest greater myogenic tone with increased calcium sensitization in SHR arteries, largely because of Rho kinase activation, with a minor contribution of PKC activation.     

Brain synaptosomal Ca2+ uptake: comparison of Sprague-Dawley, Wistar-Kyoto and spontaneously hypertensive rats

1. K(+)-stimulated 45Ca2+ uptake by synaptosomes was measured with respect to the strain differences between Sprague-Dawley (SD), Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). 2. 45Ca2+ uptake by synaptosomes isolated from cerebral cortex of SD, WKY and SHR was measured at 15, 30, 60, 120 and 240 sec time periods. 3. The sequence of both the magnitude and rate of resting and depolarization-dependent 45Ca2+ uptake was SHR greater than WKY greater than SD. 4. The fastest rates of resting and depolarization-dependent 45Ca2+ uptake occurred in each rat during the first 15 sec and uptake rates dropped off quickly in both resting and depolarization states. 5. At 15 sec, there were significant differences between SHR and WKY, while there were no significant differences between WKY and SD. 6. The results suggest that an important alteration in Ca2+ channel characteristics may occur in SHR brain synaptosomes.     

The ultraviolet studies on protein-lipid interaction of a protein kinase C-gamma phorbol-binding domain.

Family of protein kinase C (PKC) isozymes play a key role in transducing a vast number of signals into the cells. The members of classical PKC family are activated by binding of various lipid ligands to one of the several cysteine-rich domains of the enzyme. Second cysteine-rich (Cys2) domain of PKC-gamma was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST) using the cDNA sequence from rat brain. The Cys2 protein after cleavage from GST was purified to homogeneity using glutathione-agarose and Mono-S cation exchanger column. In order to investigate the interaction of lipids and calcium with Cys2 protein we used UW spectroscopy. The UV spectrum of Cys2 protein exhibited a maximum at 205 nm. Exposition of Cys2 protein to phosphatidylserine (PS) vesicles resulted in significant decrease in the absorbance in the 210 nm region. Changes in UW spectrum of Cys2 protein induced by phorbol 12,13-dibutyrate (PDB) were smaller than those induced by PS, and addition of PDB with PS had no effect on the PS induced changes in UV spectrum of Cys2. Neither phosphatidylcholine (PC) nor phosphatidylethanolamine (PE) affected UV spectrum of Cys2 but in the presence of phosphatidylinositol 4,5 bisphosphate (PIP2) or phosphatidyliinositol 4-phosphate (PIP) vesicles some changes were observed. Calcium ions alone or in the presence of PS had no effect on the UV spectrum of Cys2 protein. These data indicate that PS comparing to PDB, interacts with a larger area of Cys2 protein, and that the binding sites for these two molecules are at least overlapping. The site of PIP and PIP2 interaction with PKC-gamma is distinct from that of phorbol ester binding site.