Skip to main content
NCBI home page
Journal of Cerebral Blood Flow & Metabolism logoLink to Journal of Cerebral Blood Flow & Metabolism
. 2014 Jan 22;34(4):613–620. doi: 10.1038/jcbfm.2013.232

Protein kinase C delta modulates endothelial nitric oxide synthase after cardiac arrest

Hung Wen Lin 1,*, Victoria L Gresia 1, Holly M Stradecki 1, Aleksey Alekseyenko 1, Cameron Dezfulian 2, Jake T Neumann 1, Kunjan R Dave 1, Miguel A Perez-Pinzon 1
PMCID: PMC3982078  PMID: 24447953

Abstract

We previously showed that inhibition of protein kinase C delta (PKCδ) improves brain perfusion 24 hours after asphyxial cardiac arrest (ACA) and confers neuroprotection in the cortex and CA1 region of the hippocampus 7 days after arrest. Therefore, in this study, we investigate the mechanism of action of PKCδ-mediated hypoperfusion after ACA in the rat by using the two-photon laser scanning microscopy (TPLSM) to observe cortical cerebral blood flow (CBF) and laser Doppler flowmetry (LDF) detecting regional CBF in the presence/absence of δV1-1 (specific PKCδ inhibitor), nitric oxide synthase (NOS) substrate (L-arginine, L-arg) and inhibitor (Nω-Nitro-L-arginine, NLA), and nitric oxide (NO) donor (sodium nitroprusside, SNP). There was an increase in regional LDF and local (TPLSM) CBF in the presence of δV1-1+L-arg, but only an increase in regional CBF under δV1-1+SNP treatments. Systemic blood nitrite levels were measured 15 minutes and 24 hours after ACA. Nitrite levels were enhanced by pretreatment with δV1-1 30 minutes before ACA possibly attributable to enhanced endothelial NOS protein levels. Our results suggest that PKCδ can modulate NO machinery in cerebral vasculature. Protein kinase C delta can depress endothelial NOS blunting CBF resulting in hypoperfusion, but can be reversed with δV1-1 improving brain perfusion, thus providing subsequent neuroprotection after ACA.

Keywords: asphyxial cardiac arrest, middle cerebral artery occlusion, neuroprotection, palmitic acid methyl ester, stearic acid methyl ester.

Introduction

We previously showed that inhibition of protein kinase C delta (PKCδ) via δV1-1 can increase perfusion 24 hours after asphyxial cardiac arrest (ACA). Global cerebral ischemia (via ACA) causes derangement of cerebral blood flow (CBF) responsible for neuronal cell death in the CA1 region of the hippocampus as well as the cortex.1 Owing to the overall decrease in CBF after ischemia, neuronal cell death1 can occur in major regions of the brain responsible for learning, memory, and cognitive function.2 It is thought that during global ischemia, PKCδ (a novel PKC) levels are elevated causing PKCδ to translocate to the nucleus (activation) resulting in cellular damage. In the normal brain, PKCδ levels are nominal whereas global ischemia can cause activation/translocation of PKCδ.3 Inhibition of PKCδ (via specific inhibitor of PKCδ, δV1-1) can cause a revival of CBF 24 hours after ischemia to counteract hypoperfusion or low CBF found to be suppressed after cardiac arrest from 38% to 65%.1, 4 We previously showed that pretreatment of δV1-1 before ACA can enhance perfusion 24 hours after ischemia, resulting in improved neuronal survival in the hippocampal CA1 and cortex regions in our rat model of ACA.1 Here, we sought out to define the specific mechanism(s) of how inhibition of PKCδ can alleviate these pathologies. The possible endothelium and endothelial-mediated nitric oxide synthase (eNOS) involvement as a target for PKCδ relating to general circulation was first reported by Monti et al.5 This led to these current experiments in the brain by utilizing inhibitors and substrates related to nitric oxide synthase (NOS). Our results suggest that pretreatment with δV1-1 before ACA can activate eNOS machinery suggesting that the enhancement of perfusion may be attributed to enhanced CBF. This is further supported by the fact that L-arg (a substrate for nitric oxide (NO) production) and sodium nitroprusside (SNP, NO donor) can both enhance CBF in the nonischemic rat as measured by two-photon laser scanning microscopy (TPLSM) of cortical microvessels as well as laser Doppler flowmetry (LDF, a measure of regional CBF). Systemic whole-blood nitrite (NO metabolite) was also measured before and after ACA in the presence or absence of δV1-1. Administration of δV1-1 mediated increased perfusion, enhanced nitrite levels 24 hours after ACA as compared with control suggesting the involvement of NO modulation. In addition, only eNOS levels (as opposed to other evaluated NOS isoforms) were elevated during this process. Here, we show that inhibition of PKCδ before ACA can alleviate the detrimental effects of hypoperfusion via eNOS activation.

Materials and Methods

Chemicals

L-arginine hydrochloride (100 mg/kg), SNP (0.75 mg/kg), and Nω-Nitro-L-arginine hydrochloride (10 mg/kg) (Sigma-Aldrich, St Louis, MO, USA) were diluted in sterile saline and injected (700 μL total injection volume) into the Sprague–Dawley rat intravenously. Protein kinase C delta inhibitor (δV1-1) and tat carrier peptide (0.5 mg/kg) (KAI pharmaceuticals, San Francisco, CA, USA) were dissolved in sterile saline (0.9%) and injected into the rat intravenously (700 μL total injection volume). Each drug was administered every 30 minutes as denoted in Figures 1A and 2A.

Figure 1.

Figure 1

Open in a new tab

Schematic diagram of the experimental design. (A) Baseline two-photon laser scanning microscopy (TPLSM) measurements were acquired before any drug administration. Subsequently, L-arg (100 mg/kg) or sodium nitroprusside (SNP) (0.75 mg/kg) was injected (bolus, intravenously) into the rat. After drug injection, a linescan image was acquired via TPLSM at 5, 10, 15, and 30-minute intervals (represented with gray arrows). After administration of SNP, δV1-1 (0.5 mg/kg) was injected with a subsequent dose of L-arg (100 mg/kg) or SNP (0.75 mg/kg) administered intravenously 30 minutes after the introduction of δV1-1. Linescan image acquisition via TPLSM was implemented at 5, 10, 15, and 30-minute intervals. (B) Inhibition of protein kinase C delta (PKCδ) promoted L-arg-induced enhancement of cortical cerebral blood flow (CBF) via TPLSM. We used TPLSM to examine local CBF within the cortical microvessels (penetrating pial microvessels). Administration of L-arg (100 mg/kg) or SNP (0.75 mg/kg) alone did not produce a profound change in CBF. However, upon administration of δV1-1 (for 30 minutes), L-arg (216±48%, at t=105 minutes) but not SNP-enhanced CBF (n=9, *P⩽0.05).

Figure 2.

Figure 2

Open in a new tab

(A) The laser Doppler flowmetry (LDF) probe was placed in a similar position as the two-photon laser scanning microscopy (TPLSM) objective (1 mm lateral to the bregma). Laser Doppler flowmetry measurement of regional cerebral blood flow (CBF) was recorded for 30 minutes before the start of each experiment to obtain baseline measurements (t=0 to 30 minutes). Subsequently tat peptide or δV1-1 (0.5 mg/kg) was administered and regional CBF was recorded for 30 minutes. Subsequent bolus injections of L-arg (100 mg/kg), Nω-Nitro-L-arginine (NLA) (10 mg/kg), and sodium nitroprusside (SNP) (0.75 mg/kg) were introduced every 30 minutes with continuous LDF recordings at 2 Hz. (B) Inhibition of protein kinase C delta (PKCδ) promoted L-arg and SNP-induced enhancement of regional CBF via LDF. We used LDF to examine regional CBF. Administration of L-arg (100 mg/kg) (121±5%), NLA (10 mg/kg) (110±4%), or SNP (0.75 mg/kg) (143±8%) in the presence of tat peptide (n=7) did not produce profound changes in CBF. However, upon administration of δV1-1 (for 30 minutes, n=8), L-arg (155±13%) and SNP (188±11%) enhanced CBF (n=7 to 8, *P⩽0.05).

Animal Preparation

All procedures were approved by the Institutional Animal Care and Use Committee (University of Miami, Miller School of Medicine) and written up in accordance with ARRIVE. Adult male Sprague–Dawley rats (250 to 350 g) were fasted overnight before surgery. Rats were anesthetized with 4% isoflurane and 30:70 mixture of O2 and N2O, followed by endotracheal intubation. Isoflurane was lowered to 1.5% to 2% for endovascular access. The femoral vein and artery were cannulated using a single-lumen (PE-50) catheter for blood pressure monitoring, blood gas analysis, and intravenous injection of pharmacological agents. The rats were immobilized with vecuronium bromide (2.0 mg/kg, intravenously, administered every 10 minutes) and maintained immobilized throughout the procedure. Head and body temperatures were maintained at 37°C using heating blankets and lamps.

Thinned-Skull Window Method

The thinned-skull window method is a modified method from Xu et al.6 Anesthetized rats were placed on a stereotaxic frame to ensure the stability of the rat. After femoral arteries and vein of the rat were cannulated and carotid ligatures installed, a longitudinal incision extending from the neck region to the frontal area of the rat was made at the midline scalp using microsurgical tools. Utilizing a high-speed microdrill, a thin circular area of the skull (∼2 mm in diameter) was made 1 mm lateral to the bregma. Thinned-skull window was created carefully with a microdrill thinning out the skull with constant irrigation with sterile saline so as to not overheat the skull or the drill bit. The skull is thin enough for visualization of blood vessels via TPLSM when it is approximately half the thickness of the skull or approximately 0.5 mm.1, 6

Two-photon Laser Scanning Microscopy

After thinning the skull, the rat was placed on a TPLSM (Lasersharp2000, Bio-Rad, Hercules, CA, USA). pH, mean arterial blood pressure, pCO2, pO2, glucose, rectal, and head temperatures were constantly monitored throughout the experiment. A 20 × water immersion objective (Olympus XLUMPlanFl) was lowered in proximity to the thinned-skull window. Fluorescent images were captured (every 5 to 15 minutes) at an excitation wavelength of 910 nm with the intravenous introduction of low molecular weight fluorescein–dextran. Cortical cerebral blood vessel images were captured at 20 × and 200 × . Additionally, Z-series (20 × ) and linescan (200 × ) images were obtained throughout the time course of the experiment starting with t=0 (before the introduction of drugs). We have previously experienced no immediate change in physiologic variables or change in CBF or blood vessel diameters upon acute administration of tat peptide or δV1-1.1 Only microvessels with diameters 5 to 10 μm were considered for TPLSM studies. Upon visual inspection, arterioles can be recognized morphologically based on arterioles branching out from larger vessels, while, venules merge into larger vessels.7

Linescans for red blood cell (RBC) velocity measurements were analyzed with ImageJ analysis software.8 Each velocity measurement was calculated by measuring the slope of the dark lines (10 lines) representing 10 RBCs traversing at a point in time of the linescan. The slopes were calculated and averaged. One microvessel was studied per rat with an overall diameter on average of 10 μm. Linescan images were captured and measured as denoted in Figure 1A as ‘TPLSM measurements in normal rats'. Our data were expressed as percent change in flow. This was achieved by utilizing each rat as its own internal control. Baseline measurements before drug treatment were used for normalization of the data. This analysis was necessary owing to the fact that not all microvasculature possess exactly the same vessel size or RBC velocities before ischemia and drug treatments. Statistical analysis was evaluated by one-way ANOVA followed by Tukey's post hoc test.

Laser Doppler Flowmetry

Laser doppler flowmetry measurements were obtained to determine CBF dynamics of cortical blood vessels in rats with no treatment (baseline), tat peptide or δV1-1 (0.5 mg/kg), L-arg (100 mg/kg), NLA (10 mg/kg), and SNP (0.75 mg/kg) (all bolus with 700 μL total injection volume at 30 minute intervals, intravenously). Laser Doppler flowmetry measurements of blood perfusion were monitored at the start of the experiment and continuously recorded (2 Hz sampling frequency) (30 minutes for each drug injection) until the end of the final drug injections. To monitor blood perfusion in the cerebral cortex, a 2 mm2 burr hole was made over the left frontoparietal cortex ∼1 mm lateral to the bregma. The bone was drilled to a thin layer with a cutting burr under saline irrigation, and a cortical area with blood vessels less than 50 μm diameter was selected by visualization through the thin bone layer, and a fiber optic probe (1 mm2) was placed thereupon. The fiber optic probe, when coupled to a PeriFlux 4001 Master laser Doppler blood perfusion monitor (Perimed, Jarfalla, Sweden), measures cerebral blood perfusion in a 1 mm3 tissue region. The Doppler signals were routed to a polygraphic recording system interfaced to a personal computer, via an A to D converter, utilizing data acquisition software (Perisoft for Windows, Jarfalla, Sweden). The average of all peak values of each 30-minute drug treatment was averaged and calculated using the 2 Hz sampling rate. All data are expressed as mean±s.e.m.9 Statistical analysis was evaluated by one-way ANOVA followed by Tukey's post hoc test.

Asphyxial Cardiac Arrest

To induce ACA, apnea was induced by disconnecting the ventilator from the endotracheal tube. Six minutes after asphyxia, resuscitation was initiated by administering a bolus injection of epinephrine (0.005 mg/kg, intravenously) and sodium bicarbonate (1 mEq/kg, intravenously) followed by mechanical ventilation. Arterial blood gases were measured before and after ACA. Control animals (sham) were subjected to surgical procedures similar to ACA animals except without induction of ACA. Resuscitation drugs were not used; however, sham animals were treated with isoflurane similar to experimental animals. Based on our prior experiences, administration of epinephrine in sham animals does not have any CBF differences after the blood pressure returns to normal. The rats were immobilized with vecuronium bromide (2.0 mg/kg, intravenously, administered every 10 minutes) and maintained immobilized throughout the procedure.1

Whole-Blood Nitrite Analysis

Rat whole-blood was extracted before, 15 minutes, and 24 hours after ACA. Nitrite preservation solution was added to the whole blood. Nitrite measurements were determined by tri-iodide-based gas-phase reductive chemiluminescence with an NO analyzer (GE Analytic, Boulder, CO, USA) as described previously.10 Nitrite concentrations were calculated based on the area under the curve (peak) utilizing a known reference injection of nitrite. Statistical analysis was evaluated by one-way ANOVA followed by Tukey's post hoc test.

Western Blot Analysis

Rats were prepared as noted in the ‘animal preparation' section. Rats were injected with either tat peptide or δV1-1 and 6 minutes of ACA was performed as already mentioned. One hour after tat peptide or δV1-1 injection with or without ACA, the rats were killed and the brain excised. The cortex (the region of CBF measurements via LDF and TPLSM) was extracted and homogenized using a glass homogenizer in RIPA (50 mmol/L Tris pH 8, 150 mmol/L NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 1% SDS). The homogenate was centrifuged at 13,000 × g for 15 minutes at 4°C and the protein fractions were quantified using the Bradford Assay (Bio-Rad Dc Protein Assay, Hercules, CA, USA). Equal amounts of protein (50 μg) were separated on a 10% SDS-PAGE gel and electroblotted to nitrocellulose. Membranes were blocked with 5% bovine serum albumin/tris buffered saline with tween 20 (TBS-T) (for phospho-specific antibodies) or 5% milk/TBS-T (non-phospho-specific antibodies) then incubated in primary antibody at 4°C overnight. Primary antibodies were obtained from Cell Signaling (Boston, MA, USA; phospho-eNOS (Ser1177), nNOS) and Abcam (Cambridge, MA, USA; eNOS, iNOS). Proteins were detected by appropriate horseradish peroxidase-conjugated secondary antibodies (GE Healthcare UK Limited, Buckinghamshire, UK) and enhanced chemiluminescence (ECL) system (Pierce Thermo Scientific, Rockford, IL, USA). Protein loading was determined by re-probing the membrane for β-actin (Sigma-Aldrich). The blots were imaged and analyzed using Bio-Rad Quantity One Analysis Software. Graphical results are expressed as fold change from tat peptide normalized to β-actin. Statistical analysis was evaluated by one-way ANOVA followed by Tukey's post hoc test or Student's t-test for unpaired samples as appropriate with SPSS statistical software (Chicago, IL, USA).

Statistical Analysis

Results were expressed as means±s.e.m. Statistical analysis was evaluated by one-way ANOVA followed by Tukey's post hoc test or Student's t-test for paired or unpaired samples as appropriate with SPSS statistical software. The P⩽0.05 level of probability was accepted as significant.

Results

Protein Kinase C Delta Modulates L-Arginine-induced Enhancement of Cerebral Blood Flow in Cortical Microvessels

We previously showed that inhibition of PKCδ via δV1-1 can cause an increase in CBF 24 hours after ACA blunting hypoperfusion.1 This leads to our current hypothesis that PKCδ alone can modulate NO machinery in acute or chronic situations. We applied (see Figure 1A, experimental paradigm) L-arg (100 mg/kg) (substrate for NOS) or SNP (0.75 mg/kg) (NO donor) to determine if NO is involved in PKCδ-mediated CBF via TPLSM of cortical microvessels. Rats injected (bolus) with L-arg or SNP for 30 minutes each did not produce any overall significant increases in cortical CBF. It is important to note that upon time (t)=5 to 15 minutes of L-arg perfusion, cortical CBF was enhanced indicating the efficacy of L-arg in promoting enhanced CBF. In addition, infusion of SNP after L-arg at t=35 to 45 minutes produced a small enhancement of CBF suggestive of the actions of the NO donor.11 Furthermore, rats injected with δV1-1 (30 minutes) and then L-arg (30 minutes) resulted in enhanced peak cortical CBF (216±48%, at t=105 minutes) as compared with L-arg alone (113±11%, at t=5 minutes) (n=9) (Figure 1B). Administration of SNP after δV1-1 did not produce any significant changes in CBF. These results indicate that inhibition of PKCδ can enhance L-arg-induced enhancement of CBF suggesting that PKCδ can modulate NO in the cortical microvessels. It is also important to note that the concentrations of L-arg, NLA, and SNP, were selected by administering the drugs at a high enough dose without acute or chronic side effects (i.e. drastic lowering of systemic blood pressure). In addition, the infusion concentrations of L-arg,12, 13, 14 NLA,15, 16, 17 and SNP18, 19 were readily used by other investigators.

Protein Kinase C Delta Modulates L-arginine and Sodium Nitroprusside-induced Enhancement of Regional Cerebral Blood Flow

The usage of TPLSM to observe cortical microvessels gives a focal perspective on cortical CBF at a point in time; therefore, we also used LDF to obtain a regional perspective on CBF at a high data sampling rate of 2 Hz as opposed to TPLSM (every 5 to 15 minutes after induction of drugs) (Figure 2). The LDF probe was placed in the same position as the TPLSM objective of 1 mm lateral to the bregma. Our LDF results suggest that the introduction of δV1-1 with subsequent L-arg (100 mg/kg) administration enhanced regional CBF (155±13%) as compared with tat peptide+L-arg (121±5%). No significant difference in regional CBF was observed with the administration of δV1-1+NLA (124±8%) as compared with tat peptide+NLA (110±4%). Contrary to the TPLSM study, administration of δV1-1+SNP (188±11%) enhanced regional CBF as compared with tat peptide+SNP (143±8%) (n=7 to 8) (Figure 2B).

Whole-blood Nitrite Concentration is Enhanced via Inhibition of Protein Kinase C Delta After Cardiac Arrest

In order to determine the possible mechanism of action of PKCδ-mediated enhancement of CBF, we measured nitrite concentration in systemic whole-blood 15 minutes and 24 hours after ACA with tat peptide or δV1-1 pretreated for 30 minutes before the induction of ACA. Animals pretreated with δV1-1 presented with an enhanced nitrite concentration in systemic whole blood (2.50±0.61 μmol/L) 24 hours after ACA as compared with Sham, ACA only, or tat peptide (vehicle)+ACA (Figure 3). Physiologic parameters are provided in Table 1. PO2 for all groups (after ACA) were greater than 150 mm Hg owing to 100% delivery of oxygen and increased ventilator rate (60 to 80 breaths per minute) during and after resuscitation. As nitrite is a metabolite of NO,20 it is suggestive that NO was the mostly likely cause of the enhanced CBF 24 hours after ACA as previously shown1 with TPLSM.

Figure 3.

Figure 3

Open in a new tab

Inhibition of protein kinase C delta (PKCδ) enhanced the concentration of whole-blood nitrite 24 hours after asphyxial cardiac arrest (ACA). Whole-blood nitrite analyses were performed 15 minutes and 24 hours after ACA in the presence of sham (sham surgery=no ACA), ACA only, tat peptide (vehicle, treatment 30 minutes before ACA)+ACA, or δV1-1 (0.5 mg/kg, treatment 30 minutes before ACA)+ACA. Rats subjected to whole-blood nitrite analyses 24 hours after ACA in the presence of δV1-1 presented with an increase in whole-blood nitrite concentration of 2.50±0.61 μmol/L as compared with levels of nitrite 1 μmol/L or lower (see sham, ACA, and tat peptide (vehicle)+ACA) (n=6 to 8, *P⩽0.05).

Table 1. Asphyxial cardiac arrest physiologic parameters from nitrite analyses.

Group Variable Before ACA After ACA
Sham (n=6) pH 7.481±0.026 7.437±0.024
  PCO2 (mm Hg) 33.4±1.6 37.8±1.2
  PO2 (mm Hg) 132±11 174±25
  MABP (mm Hg) 106±6 101±6
  Blood glucose (mg/dL) 112±15 —
ACA (n=8) pH 7.476±0.030 7.434±0.024
  PCO2 (mm Hg) 35.8±0.6 36.0±1.4
  PO2 (mm Hg) 113±8 333±32
  MABP (mm Hg) 100±4 100±4
  Blood glucose (mg/dL) 110±9 —
tat+ACA pH 7.467±0.017 7.468±0.0351
(n=6) PCO2 (mm Hg) 34.8±1.7 36.1±3.2
  PO2 (mm Hg) 117±9 301±66
  MABP (mm Hg) 97±4 99±2
  Blood glucose (mg/dL) 116±11 —
δV1-1+ACA pH 7.426±0.015 7.411±0.031
(n=7) PCO2 (mm Hg) 34.9±1.3 37.7±2.1
  PO2 (mm Hg) 111±4 340±49
  MABP (mm Hg) 88±3 98±4
  Blood glucose (mg/dL) 99±7 —
Open in a new tab

ACA, asphyxial cardiac arrest; MABP, mean arterial blood pressure;

Physiologic parameters such as pH, PCO2, PO2, MABP, and blood glucose were measured to ensure consistency of the animals within and between groups. PO2 for all groups (after ACA) were greater than 150 mm Hg owing to 100% delivery of oxygen and increased ventilator rate (60 breaths per minute to 80 breaths per minute) during and after resuscitation. Values are mean±s.e.m.

Endothelial Nitric Oxide Synthase is Enhanced via Inhibition of Protein Kinase C Delta After Cardiac Arrest

As nitrite concentrations were enhanced in the presence of PKCδ inhibitor (via δV1-1), we performed protein analyses via Western blot for eNOS, phosphorylated-eNOS (P-eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) in the presence of tat peptide or δV1-1 (administered 30 minutes before ACA). Cortex samples (to undergo Western blot analyses, 1 mm lateral to the bregma) were isolated after the animal was killed 24 hours after ACA. Endothelial-mediated nitric oxide synthase but not P-eNOS (Figure 4A), iNOS (Figures 4C and 4D), or nNOS (Figures 4E and 4F) was enhanced in the presence of δV1-1+ACA (1.29±0.12%) as compared with tat peptide+ACA (1.00±0.08%) or δV1-1 only (0.95±0.01%) (without ACA) (Figure 4B). Physiologic parameters are provided in Table 2. PO2 for all groups (after ACA) were greater than 150 mm Hg owing to 100% delivery of oxygen and increased ventilator rate (60 to 80 breaths per minute) during and after resuscitation. These results suggest that inhibition of PKCδ can enhance eNOS levels responsible for vasodilation of cerebral arteries resulting in enhanced CBF 24 hours after ACA.

Figure 4.

Figure 4

Open in a new tab

Inhibition of protein kinase C (PKCδ) enhanced endothelial-mediated nitric oxide synthase (eNOS) but not inducible NOS (iNOS) or neuronal NOS (nNOS) cortical protein expression 24 hours after asphyxial cardiac arrest (ACA). (A) Inhibition of PKCδ (via δV1-1) pretreatment 30 minutes before ACA enhanced eNOS (δV1-1+ACA, 1.29±0.12%) but not P(Ser1177)-eNOS protein expression 24 hours as compared with tat peptide (1.00±0.08%) after ACA (B). Pretreatment with δV1-1 for 1 hour in the absence of ACA did not change eNOS protein expression (0.95±0.01%) as compared with tat peptide+ACA (B). Pretreatment with δV1-1 or tat peptide+ACA did not change iNOS or nNOS protein expression (C–F). Parentheses inside the bar graphs represent the number of experiments performed. β-actin was used as an internal loading control in all blots (*P⩽0.05).

Table 2. Physiologic parameters from Western blot analyses.

Group Variable Before ACA After ACA
tat+ACA pH 7.460±0.016 7.429±0.025
  PCO2 (mm Hg) 35.83±1.06 38.2±2.19
  PO2 (mm Hg) 129±12 299±53
  MABP (mm Hg) 103±7 107±6
  Blood glucose (mg/dL) 125±10 —
δV1-1 pH 7.470±0.014 7.436±0.013
+ACA PCO2 (mm Hg) 35.1±1.8 37.8±2.6
  PO2 (mm Hg) 106±6 249±66
  MABP (mm Hg) 99±4 103±3
  Blood glucose (mg/dL) 144±21 —
Open in a new tab

ACA, asphyxial cardiac arrest; MABP, mean arterial blood pressure.

Physiologic parameters such as pH, PCO2, PO2, MABP, and blood glucose were measured to ensure consistency of the animals within and between groups. PO2 for all groups (after ACA) were greater than 150 mm Hg owing to 100% delivery of oxygen and increased ventilator rate (60 breaths per minute to 80 breaths per minute) during and after resuscitation. Values are mean±s.e.m.

Discussion

Previously, we showed that inhibition of PKCδ via δV1-1 24 hours after ACA enhanced perfusion as compared with tat peptide pretreatment.1 This led to subsequent studies aimed at defining how inhibition of PKCδ is enhancing brain perfusion 24 hours after ACA. In this current study, we identified that PKCδ targets directly or indirectly the NO machinery evidenced by enhanced L-arg and/or SNP-induced CBF by TPLSM and LDF. In addition, inhibition of PKCδ enhanced whole-blood nitrite concentration 24 hours after ACA suggesting that the attenuation via δV1-1-induced hypoperfusion 24 hours after ACA as shown previously1 is most likely mediated by NO owing to the fact that nitrite is a major NO metabolite.20 Furthermore, we show that there is a correlative elevation in eNOS levels.

It is without a doubt that SNP has been denoted as a NO donor causing vasodilation in cerebral as well as systemic arteries. Current dogma has led us to believe that this vasodilation can cause the possibility of increased CBF.21, 22, 23 However, there are many reports that suggest otherwise.24, 25, 26 Sodium nitroprusside-mediated enhancement of CBF depends upon where and what type(s) of vasculature one is measuring from. Traditionally, studies involving ex vivo manipulation of cerebral or systemic vessel preparation has been used to study these types of phenomena utilizing pharmacological manipulations (i.e. in the presence or absence of SNP/NLA etc.) to determine vasodilation or vasoconstriction of cerebral vessels but it does not always directly correlate to enhanced CBF. In this study, we are not claiming vascular tonicity but only CBF perfusion in two techniques employed, the TPLSM of pial circulation and LDF. The objective (from TPLSM) or probe (from LDF) was placed 1 mm lateral to the bregma as published earlier.1, 27 These two techniques are real-time CBF measurements to determine the effects of PKCδ. We used L-arg, SNP, and NLA to define the relationship of PKCδ and NOS machinery.

The therapeutic potential via the modulation of PKCδ varies from vascular restenosis28 to improvement of endothelial vascular dysfunction via inhibition of PKCδ.5 It is thought that the inhibition of PKCδ (via δV1-1) can have beneficial effects against ischemia such as attenuation of hyperemia and hypoperfusion in models of global ischemia1 and vascular protection against endothelial vascular dysfunction via modulation of eNOS.5 In addition, degradation of PKCδ during reperfusion improves physiologic outcome in the myocardial infarction29 model altogether suggesting that ischemia causes activation/translocation of PKCδ whereas inhibition or attenuation of PKCδ can improve physiologic outcomes, as it relates to neuronal protection and/or revival of CBF.1, 27

We and others have reported that inhibition of PKCδ via δV1-1 can restore brain blood flow in global or regional ischemia,1, 27, 30 but the mechanism of action remains controversial. In fact, Monti et al5 suggest in models of cell culture that the effects of δV1-1 in endothelial cells can be complemented with the action of PKCɛ to modulate and stabilize eNOS preventing endothelial cell dysfunction. In addition, inhibition of PKCδ can enrich endothelial cell survival under hypoxic conditions in human umbilical vein endothelial cells.31 In coronary postcapillary venular endothelial cells, inhibition of PKCδ with δV1-1 shifts PKCɛ to the cytoplasm and PKCɛ can then translocate to the membrane. Protein kinase C ɛ activation promotes inhibition of eNOS resulting in decreased reactive oxygen species production via Akt phosphorylation.5 This is contrary to our results. However, using a model of bovine aortic endothelial cells, Rask-Madsen and King,32 suggested that PKCɛ can activate Akt and eNOS through the vascular endothelial growth factor pathway.32 Together, these results from Monti et al5 and Rask-Madsen and King32 can provide a possible explanation of our results where inhibition of PKCδ via δV1-1 can activate PKCɛ translocation causing enhanced eNOS expression. It is also important to note that although not without controversy, many of these studies were performed in cell culture systems using methods to mimic ischemia, such as serum deprivation. As cardiac arrest is a multi-faceted problem consisting of whole-body ischemia that compromises systemic blood parameters and cerebral, renal, and cardiac functions, consequent disruption of CBF, it is possible that our results can provide new additional insight into this problem.

We initially used the TPLSM to detect changes in cortical CBF in the presence of L-arg and SNP. We observed L-arg-induced enhancement of CBF in pial circulation using TPLSM in the presence of PKCδ inhibitor (δV1-1). As TPLSM technically measures RBC speed in pial circulation, we implemented LDF by observing regional CBF at the same location as TPLSM. Measuring RBC speed confirms that the effects observed via TPLSM were similar in the presence of NO substrates with the exception of SNP-induced enhancement of CBF in the presence of δV1-1 (Figures 1 and 2). This discrepancy (SNP-induced relaxation between TPLSM and LDF methods) between the two methods may be attributed to the limitation of the TPLSM acquiring an image at ∼5-minute intervals to examine peak CBF per image, whereas LDF (probe placed 1 mm lateral to the bregma, similar to the acquisition of images via TPLSM) samples at a rate of 2 Hz to obtain the average of peak values during the drug application(s) (bolus intravenous injection of L-arg or SNP). We recognize that TPLSM is used to detect microvessel blood flow (⩽20 μm in diameter) whereas the use of LDF (using different technologies) detects overall CBF providing a more broad perspective (1 mm3) as to the overall status of CBF in the presence of PKCδ.

With the use of L-arg, NLA or SNP as a pharmacological method to detect the intricate involvement of NO in TPLSM and LDF experiments (Figures 1 and 2), our data further support a major role of NO-mediated vasoactive modulation in the presence of PKCδ. Our initial findings (Figure 3) measuring nitrite (NO metabolite) concentrations in whole blood before and after ACA in the presence or absence of δV1-1 suggest that whole-blood nitrite concentrations are elevated in the presence of δV1-1 24 hours after ACA as compared with sham, ACA, or tat peptide+ACA. In a separate set of experiments, Western blot analyses (Figure 4) in whole cortical brain tissue suggest that the levels of eNOS but not phospho-eNOS were enhanced in the presence of ACA pretreated with δV1-1. Based on our overall evidence, it is suggestive that the nitrite (NO metabolite) from whole-blood could be derived from eNOS as correlated in our Western blot studies. However, we cannot rule out the possibility that the contributions of whole-blood nitrite can originate from iNOS, nNOS, or hemoglobin.33 These results suggest that perhaps eNOS activation does not solely require phosphorylation in vivo unlike conventional NO activation pathways mediated by eNOS phosphorylation at serine 1177 or 1179.34, 35, 36 Traditionally, phosphorylation sites at serine 1177/117936 causes eNOS activation while serine 495 is thought to be inhibitory.37 The main hypothesis and the actual experimental design is addressing the inhibition of PKCδ (via δV1-1) enhances eNOS after ACA as suggested in Figures 3 and 4. We did not study S495 because when phosphorylated, inhibits eNOS. As the inhibition of PKCδ enhances eNOS and theoretically decreases in phosphorylation, we focused on the activation site of eNOS.

Much of the current understanding regarding the mechanism of activation of NOS has been performed in intact cell cultures suggesting that perhaps there may be differential/additional factors (such as Ca2+ levels responsible for the modulation in various NOS systems) that mediate NOS activation. Others have suggested that the relationship between PKCδ and eNOS activation may be tissue/cell specific.5 Previously, others have shown that PKCδ phosphorylates serine (1177/1179) resulting in eNOS activation in bovine aortic endothelial cells.38 Attenuation of PKCδ (via small interfering RNA) decreased NO production in fibroblasts.39 In both of these studies, the authors used Rottlerin, which at the time was thought to be the only specific inhibitor of PKCδ but others have shown that Rottlerin is not specific for PKCδ.40 Nonetheless, this leads to our novel findings, which suggest that inhibition of PKCδ (via δV1-1) actually enhances whole-blood nitrite concentration (Figure 3) with eNOS-enhanced levels in the presence of ACA pretreated with δV1-1 (Figure 4). In addition, some investigators have suggested that enhanced P-eNOS in turn, inhibit PKCδ expression in human umbilical vein endothelial cells induced with propofol41 whereas Ramzy et al42 suggested that decreased eNOS expression results in increased PKCδ levels in human saphenous vein endothelial cells induced with endothelin-1.42 Therefore, based on this information from Ramzy et al,42 inhibition of PKCδ will increase eNOS levels similar to our findings (Figure 4).

In summary, we show that inhibition of PKCδ via δV1-1 can enhance brain perfusion 24 hours after ACA.1 This enhancement in CBF is most likely mediated via enhanced eNOS levels in the presence of PKC suppression. This is further evidenced by enhanced nitrite concentrations in whole-blood 24 hours after ACA in the presence of δV1-1. More studies are needed to define the physiologic role of eNOS in the context of cardiac arrest beyond activation via phosphorylation as well as defining the therapeutic value of modulation of PKCδ (via δV1-1) against cerebral ischemia.

The authors declare no conflict of interest.

Footnotes

This work was supported by National Institutes of Health grants NS45676-01, NS054147-01, NS34773, NS073779, American Heart Association-Philips grant 10POST4340011, and AHA-13SDG13950014.

References

  1. Lin HW, Defazio RA, Della-Morte D, Thompson JW, Narayanan SV, Raval AP, et al. Derangements of post-ischemic cerebral blood flow by protein kinase C delta. Neuroscience. 2010;171:566–576. doi: 10.1016/j.neuroscience.2010.08.058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alexander MP, Lafleche G, Schnyer D, Lim C, Verfaellie M. Cognitive and functional outcome after out of hospital cardiac arrest. J Int Neuropsychol Soc. 2011;17:364–368. doi: 10.1017/S1355617710001633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Raval AP, Dave KR, Prado R, Katz LM, Busto R, Sick TJ, et al. Protein kinase C delta cleavage initiates an aberrant signal transduction pathway after cardiac arrest and oxygen glucose deprivation. J Cereb Blood Flow Metab. 2005;25:730–741. doi: 10.1038/sj.jcbfm.9600071. [DOI] [PubMed] [Google Scholar]
  4. Manole MD, Foley LM, Hitchens TK, Kochanek PM, Hickey RW, Bayir H, et al. Magnetic resonance imaging assessment of regional cerebral blood flow after asphyxial cardiac arrest in immature rats. J Cereb Blood Flow Metab. 2009;29:197–205. doi: 10.1038/jcbfm.2008.112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Monti M, Donnini S, Giachetti A, Mochly-Rosen D, Ziche M. deltaPKC inhibition or varepsilonPKC activation repairs endothelial vascular dysfunction by regulating eNOS post-translational modification. J Mol Cell Cardiol. 2010;48:746–756. doi: 10.1016/j.yjmcc.2009.11.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Xu HT, Pan F, Yang G, Gan WB. Choice of cranial window type for in vivo imaging affects dendritic spine turnover in the cortex. Nat Neurosci. 2007;10:549–551. doi: 10.1038/nn1883. [DOI] [PubMed] [Google Scholar]
  7. Koike N, Fukumura D, Gralla O, Au P, Schechner JS, Jain RK. Tissue engineering: creation of long-lasting blood vessels. Nature. 2004;428:138–139. doi: 10.1038/428138a. [DOI] [PubMed] [Google Scholar]
  8. Nishimura N, Schaffer CB, Friedman B, Lyden PD, Kleinfeld D. Penetrating arterioles are a bottleneck in the perfusion of neocortex. Proc Natl Acad Sci USA. 2007;104:365–370. doi: 10.1073/pnas.0609551104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Della-Morte D, Raval AP, Dave KR, Lin HW, Perez-Pinzon MA. Post-ischemic activation of protein kinase C epsilon protects the hippocampus from cerebral ischemic injury via alterations in cerebral blood flow. Neurosci Lett. 2011;487:158–162. doi: 10.1016/j.neulet.2010.10.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dezfulian C, Shiva S, Alekseyenko A, Pendyal A, Beiser DG, Munasinghe JP, et al. Nitrite therapy after cardiac arrest reduces reactive oxygen species generation, improves cardiac and neurological function, and enhances survival via reversible inhibition of mitochondrial complex I. Circulation. 2009;120:897–905. doi: 10.1161/CIRCULATIONAHA.109.853267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Schumann-Bard P, Touzani O, Young AR, Toutain J, Baron JC, Mackenzie ET, et al. Cerebrovascular effects of sodium nitroprusside in the anaesthetized baboon: a positron emission tomographic study. J Cereb Blood Flow Metab. 2005;25:535–544. doi: 10.1038/sj.jcbfm.9600044. [DOI] [PubMed] [Google Scholar]
  12. Gray GA, Schott C, Julou-Schaeffer G, Fleming I, Parratt JR, Stoclet JC. The effect of inhibitors of the L-arginine/nitric oxide pathway on endotoxin-induced loss of vascular responsiveness in anaesthetized rats. Br J Pharmacol. 1991;103:1218–1224. doi: 10.1111/j.1476-5381.1991.tb12327.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Julou-Schaeffer G, Gray GA, Fleming I, Schott C, Parratt JR, Stoclet JC. Loss of vascular responsiveness induced by endotoxin involves L-arginine pathway. Am J Physiol. 1990;259:H1038–H1043. doi: 10.1152/ajpheart.1990.259.4.H1038. [DOI] [PubMed] [Google Scholar]
  14. Gardiner SM, Compton AM, Kemp PA, Bennett T. Regional and cardiac haemodynamic effects of NG-nitro-L-arginine methyl ester in conscious, Long Evans rats. Br J Pharmacol. 1990;101:625–631. doi: 10.1111/j.1476-5381.1990.tb14131.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dorrepaal CA, Shadid M, Steendijk P, Van der Velde ET, Van de Bor M, Baan J, et al. Effect of post-hypoxic-ischemic inhibition of nitric oxide synthesis on cerebral blood flow, metabolism and electrocortical brain activity in newborn lambs. Biol Neonate. 1997;72:216–226. doi: 10.1159/000244487. [DOI] [PubMed] [Google Scholar]
  16. Piotrovskij V, Kallay Z, Horecky J, Trnovec T, Krumpl G, Krejcy K. Dose-ranging study of NG-nitro-L-arginine pharmacokinetics in rats after bolus intravenous administration. Xenobiotica. 1994;24:663–669. doi: 10.3109/00498259409043268. [DOI] [PubMed] [Google Scholar]
  17. Traystman RJ, Moore LE, Helfaer MA, Davis S, Banasiak K, Williams M, et al. Nitro-L-arginine analogues. Dose- and time-related nitric oxide synthase inhibition in brain. Stroke. 1995;26:864–869. doi: 10.1161/01.str.26.5.864. [DOI] [PubMed] [Google Scholar]
  18. Kurihara N, Yanagisawa H, Sato M, Tien CK, Wada O. Increased renal vascular resistance in zinc-deficient rats: role of nitric oxide and superoxide. Clin Exp Pharmacol Physiol. 2002;29:1096–1104. doi: 10.1046/j.1440-1681.2002.03783.x. [DOI] [PubMed] [Google Scholar]
  19. Hobel M, Kreye VA, Nemes Z, Pill J. Organotoxic effects of excessive doses of sodium nitroprusside in the rabbit. Klin Wochenschr. 1978;56 (Suppl 1:153–159. doi: 10.1007/BF01477467. [DOI] [PubMed] [Google Scholar]
  20. Tsikas D. Methods of quantitative analysis of the nitric oxide metabolites nitrite and nitrate in human biological fluids. Free Radic Res. 2005;39:797–815. doi: 10.1080/10715760500053651. [DOI] [PubMed] [Google Scholar]
  21. Candia GJ, Heros RC, Lavyne MH, Zervas NT, Nelson CN. Effect of intravenous sodium nitroprusside on cerebral blood flow and intracranial pressure. Neurosurgery. 1978;3:50–53. doi: 10.1227/00006123-197807000-00008. [DOI] [PubMed] [Google Scholar]
  22. Fitch W, Pickard JD, Tamura A, Graham DI. Effects of hypotension induced with sodium nitroprusside on the cerebral circulation before, and one week after, the subarachnoid injection of blood. J Neurol Neurosurg Psychiatry. 1988;51:88–93. doi: 10.1136/jnnp.51.1.88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hartmann A, Buttinger C, Rommel T, Czernicki Z, Trtinjiak F. Alteration of intracranial pressure, cerebral blood flow, autoregulation and carbondioxide-reactivity by hypotensive agents in baboons with intracranial hypertension. Neurochirurgia. 1989;32:37–43. doi: 10.1055/s-2008-1053998. [DOI] [PubMed] [Google Scholar]
  24. Fitch W, Ferguson GG, Sengupta D, Garibi J, Harper AM. Autoregulation of cerebral blood flow during controlled hypotension in baboons. J Neurol Neurosurg Psychiatry. 1976;39:1014–1022. doi: 10.1136/jnnp.39.10.1014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Akerman M, Arfel G, de Pommery J, Arrouvel C, Sirvys A, Vourc'h G. Experimental study of the cerebrovascular effects of sodium nitroprusside in the baboon Papio papio. Its action on the experimental vasospasm. Neurochirurgie. 1976;22:43–57. [PubMed] [Google Scholar]
  26. Sivarajan M, Amory DW, McKenzie SM. Regional blood flows during induced hypotension produced by nitroprusside or trimethaphan in the rhesus monkey. Anesth Analg. 1985;64:759–766. [PubMed] [Google Scholar]
  27. Lin HW, Della-Morte D, Thompson JW, Gresia VL, Narayanan SV, Defazio RA, et al. Differential effects of delta and epsilon protein kinase C in modulation of postischemic cerebral blood flow. Adv Exp Med Biol. 2012;737:63–69. doi: 10.1007/978-1-4614-1566-4_10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ding RQ, Tsao J, Chai H, Mochly-Rosen D, Zhou W. Therapeutic potential for protein kinase C inhibitor in vascular restenosis. J Cardiovasc Pharmacol Ther. 2011;16:160–167. doi: 10.1177/1074248410382106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Churchill EN, Ferreira JC, Brum PC, Szweda LI, Mochly-Rosen D. Ischaemic preconditioning improves proteasomal activity and increases the degradation of deltaPKC during reperfusion. Cardiovasc Res. 2010;85:385–394. doi: 10.1093/cvr/cvp334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ikeno F, Inagaki K, Rezaee M, Mochly-Rosen D. Impaired perfusion after myocardial infarction is due to reperfusion-induced deltaPKC-mediated myocardial damage. Cardiovasc Res. 2007;73:699–709. doi: 10.1016/j.cardiores.2006.12.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Shizukuda Y, Tang S, Yokota R, Ware JA. Vascular endothelial growth factor-induced endothelial cell migration and proliferation depend on a nitric oxide-mediated decrease in protein kinase Cdelta activity. Circ Res. 1999;85:247–256. doi: 10.1161/01.res.85.3.247. [DOI] [PubMed] [Google Scholar]
  32. Rask-Madsen C, King GL. Differential regulation of VEGF signaling by PKC-alpha and PKC-epsilon in endothelial cells. Arterioscler Thromb Vasc Biol. 2008;28:919–924. doi: 10.1161/ATVBAHA.108.162842. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Patel RP, Hogg N, Kim-Shapiro DB. The potential role of the red blood cell in nitrite-dependent regulation of blood flow. Cardiovasc Res. 2011;89:507–515. doi: 10.1093/cvr/cvq323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Fulton D, Gratton JP, Sessa WC. Post-translational control of endothelial nitric oxide synthase: why isn't calcium/calmodulin enough. J Pharmacol Exp Ther. 2001;299:818–824. [PubMed] [Google Scholar]
  35. Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature. 1999;399:601–605. doi: 10.1038/21224. [DOI] [PubMed] [Google Scholar]
  36. Fulton D, Gratton JP, McCabe TJ, Fontana J, Fujio Y, Walsh K, et al. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature. 1999;399:597–601. doi: 10.1038/21218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Bird IM. Endothelial nitric oxide synthase activation and nitric oxide function: new light through old windows. J Endocrinol. 2011;210:239–241. doi: 10.1530/JOE-11-0216. [DOI] [PubMed] [Google Scholar]
  38. Motley ED, Eguchi K, Patterson MM, Palmer PD, Suzuki H, Eguchi S. Mechanism of endothelial nitric oxide synthase phosphorylation and activation by thrombin. Hypertension. 2007;49:577–583. doi: 10.1161/01.HYP.0000255954.80025.34. [DOI] [PubMed] [Google Scholar]
  39. Leppanen T, Korhonen R, Laavola M, Nieminen R, Tuominen RK, Moilanen E. Down-regulation of protein kinase Cdelta inhibits inducible nitric oxide synthase expression through IRF1. PloS ONE. 2013;8:e52741. doi: 10.1371/journal.pone.0052741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Soltoff SP. Rottlerin: an inappropriate and ineffective inhibitor of PKCdelta. Trends Pharmacol Sci. 2007;28:453–458. doi: 10.1016/j.tips.2007.07.003. [DOI] [PubMed] [Google Scholar]
  41. Wang L, Jiang W. Propofol induces endothelial nitric oxide synthase phosphorylation and activation in human umbilical vein endothelial cells by inhibiting protein kinase C delta expression. Eur J Anaesthesiol. 2010;27:258–264. doi: 10.1097/EJA.0b013e3283311193. [DOI] [PubMed] [Google Scholar]
  42. Ramzy D, Rao V, Tumiati LC, Xu N, Sheshgiri R, Miriuka S, et al. Elevated endothelin-1 levels impair nitric oxide homeostasis through a PKC-dependent pathway. Circulation. 2006;114:I319–I326. doi: 10.1161/CIRCULATIONAHA.105.001503. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Cerebral Blood Flow & Metabolism are provided here courtesy of SAGE Publications

RESOURCES