Dependence of acetylcholine and ADP dilation of pial arterioles on heme oxygenase after transfusion of cell-free polymeric hemoglobin

Abstract

Polymers of cell-free hemoglobin have been designed for clinical use as oxygen carriers, but limited information is available regarding their effects on vascular regulation. We tested the hypothesis that the contribution of heme oxygenase (HO) to acetylcholine-evoked dilation of pial arterioles is upregulated 2 days after polymeric hemoglobin transfusion. Dilator responses to acetylcholine measured by intravital microscopy in anesthetized cats were blocked by superfusion of the HO inhibitor tin protoporphyrin-IX (SnPPIX) in a group that had undergone exchange transfusion with hemoglobin 2 days earlier but not in surgical sham and albumin-transfused groups. However, immunoblots from cortical brain homogenates did not reveal changes in expression of the inducible isoform HO1 or the constitutive isoform HO2 in the hemoglobin-transfused group. To test whether the inhibitory effect of SnPPIX was present acutely after hemoglobin transfusion, responses were measured within an hour of completion of the exchange transfusion. In control and albumin-transfused groups, acetylcholine responses were unaffected by SnPPIX but were blocked by addition of the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine (l-NNA) to the superfusate. In hemoglobin-transfused groups, the acetylcholine response was blocked by either SnPPIX or l-NNA alone. The effect of another HO inhibitor, chromium mesoporphyrin (CrMP), was tested on ADP, another endothelial-dependent dilator, in anesthetized rats. Pial arteriolar dilation to ADP was unaffected by CrMP in controls but was attenuated 62% by CrMP in rats transfused with hemoglobin. It is concluded that 1) polymeric hemoglobin transfusion acutely upregulates the contribution of HO to acetylcholine-induced dilation of pial arterioles in cats, 2) this upregulation persists 2 days after transfusion when 95% of the hemoglobin is cleared from the circulation, and 3) this acute upregulation of HO signaling is ubiquitous in that similar effects were observed with a different endothelial-dependent agonist (i.e., ADP) in a another species (rat).

Fig. 1

Percent change in diameter of small, intermediate, and large pial arterioles in a time-control group (n = 8 animals) and 15 min after completion of an exchange transfusion with albumin (n = 8 animals) or polymeric hemoglobin (Hb) solutions. Hb group A (n = 7 animals) subsequently was treated with tin protporphyrin-IX (SnPPIX) first. Hb group B (n = 5 animals) subsequently was treated with N^ω-nitro- L-arginine (L-NNA) first. Values are means ± SE. *P < 0.05 from time-control group.

Fig. 2

Percent change in diameter of small (A), intermediate (B), and large (C) pial arterioles after superfusion of 10 or 30 μM acetylcholine (ACh) in the acute transfusion experiment. Left, ACh responses were tested at baseline, after 10 μM SnPPIX, and after 300 μM L-NNA + 10 μM SnPPIX in a time-control group (n = 8) and in groups transfused with albumin (n = 8 animals) and Hb solutions (Gr A; n = 7 animals). Right, ACh responses were tested at baseline, after 300 μM L-NNA, and after 300 μM L-NNA + 10 μM SnPPIX in a group transfused with Hb solution (Gr B; n = 5 animals). Values are means ± SE. *P < 0.05 from time-control group at the corresponding ACh dose and number of ACh exposures.

Fig. 3

Percent change in diameter of small, intermediate, and large pial arterioles after superfusion of 10 μM 2-chloroadenosine in the acute (A) and chronic (B) transfusion experiments. Dilator responses were tested at the start and end of the experiment (after 300 μM L-NNA + 10 μM SnPPIX superfusion) in the acute and chronic control groups and groups transfused acutely or 2 days earlier with albumin and Hb solutions. In the acute experiment, Hb group A was treated with SnPPIX first, and Hb group B was first treated with L-NNA before combined treatment. Values are means ± SE.

Fig. 4

Percent change in diameter of small (A), intermediate (B), and large (C) pial arterioles after superfusion of 10 or 30 μM ACh in the chronic transfusion experiment. ACh responses were tested at baseline, after 10 μM SnPPIX, and after 300 μM L-NNA + 10 μM SnPPIX in a surgical sham-control group (n = 8 animals) and groups transfused with albumin (n = 8 animals) and Hb solutions (n = 8 animals) 2 days before the experiment. Values are means ± SE. *P < 0.05 from control group at the corresponding ACh dose and number of ACh exposures.

Fig. 5

Percent change in diameter of small, intermediate, and large pial arterioles after superfusion of 10 μM pinacidil at the end of the experiment after 300 μM L-NNA + 10 μM SnPPIX superfusion in the chronic surgical sham-control group and groups transfused 2 days earlier with albumin and Hb solutions. Values are means ± SE; n = 8 animals per group.

Fig. 6

Protein expression of heme oxygenase-1 (HO1), heme oxygenase-2 (HO2), and endothelial nitric oxide synthase (eNOS) in parietal cortex contralateral to cranial window. Optical density of the immunoblot bands (mean ± SE) is expressed as a percentage of the average value of the control group. A: expression in tissue obtained at the end of the acute transfusion experiment in control group (n = 5; 3 naïve and 2 time-control cats), albumin-transfused group (n = 5), and Hb-transfused group (n = 6). B: expression in tissue obtained at the end of the chronic transfusion experiment (2 days after transfusion) in control group (n = 7; 3 naïve and 4 surgical sham-control cats), albumin-transfused group (n = 5), and Hb-transfused group (n = 4). Analysis of variance did not indicate a significant effect among groups for any of the proteins.

Fig. 7

Pial arteriolar diameter responses (means ± SE of percent change) to 100 μM ADP superfusion before (predrug) and after (postdrug) superfusion of chromium mespoprphyrin (CrMP) in groups without Hb transfusion (n = 8 animals) and with Hb transfusion (n = 8 animals), before and after superfusion of vehicle in a group with Hb transfusion (n = 7 animals), and before and after superfusion of L-NNA in a group with Hb transfusion (n = 8 animals). *P < 0.05 from predrug value.