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

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 (N(ω)-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.

Figure 1

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

(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), N^ω-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).

Figure 3

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).

Figure 4

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).