G. L. Stahl

Activation of ischemia- and reperfusion-sensitive abdominal visceral C fiber afferents. Role of hydrogen peroxide and hydroxyl radicals.

Abdominal ischemia and reperfusion evoke reflex excitation of the cardiovascular system and generate reactive oxygen species. We have shown previously that the reactive oxygen species hydrogen peroxide (H2O2) elicits reflex excitation of the cardiovascular system after serosal application to abdominal organs. However, it is not known if ischemia-sensitive afferents respond to reactive oxygen species or if scavengers such as dimethylthiourea (DMTU) inhibit the response of these afferents to ischemia or reperfusion. Therefore, to provide more information on the neurophysiological mechanisms underlying the activation of these afferents, we studied their responses to H2O2 applied to the receptive field during recordings of single-unit activity of ischemia-insensitive or -sensitive abdominal visceral C fiber afferents in anesthetized cats. Additionally, we recorded single-unit activity of ischemia and reperfusion-sensitive afferents before and after treatment with DMTU (10 mg/kg), which scavenges H2O2 and hydroxyl radicals or the iron chelator deferoxamine (DEF, 10 mg/kg), which inhibits hydroxyl radical formation. Application of 44 mumol H2O2 to afferent endings increased the discharge frequency in nine of 11 ischemia-sensitive units, from 0.01 +/- 0.01 to 0.67 +/- 0.16 impulses per second. In contrast, only one of 10 ischemia-insensitive C fibers responded to H2O2 application. In an additional 13 ischemia-sensitive C fibers, DMTU significantly (p < 0.05) attenuated ischemia-induced increases in discharge frequency from 0.42 +/- 0.18 to 0.24 +/- 0.1 impulses per second (ischemia versus DMTU + ischemia, respectively). In eight additional C fibers, we found that reperfusion after 5 minutes of ischemia was associated with an increase in discharge activity from a baseline activity of 0.02 +/- 0.01 to 0.44 +/- 0.07 impulses per second. DMTU significantly attenuated the reperfusion-induced increases in discharge frequency from 0.08 +/- 0.04 to 0.18 +/- 0.06 impulses per second. DEF significantly (p < 0.05) attenuated the increased discharge activity from 0.39 +/- 0.07 to 0.10 +/- 0.04 impulses per second (ischemia versus DEF + ischemia, respectively) in an additional 11 ischemia-sensitive C fibers. In contrast, iron-saturated DEF did not attenuate ischemia- and reperfusion-induced increases in impulse activity. Thus, ischemia-sensitive but not ischemia-insensitive abdominal visceral afferents respond to H2O2. Furthermore, ischemia- and reperfusion-sensitive afferents decreased their impulse activity to a repeated period of ischemia or reperfusion after DMTU or DEF treatment. These data suggest that reactive oxygen species, particularly H2O2 and hydroxyl radicals, activate abdominal visceral C fibers in the cat during brief periods of ischemia and reperfusion.

In vivo and in vitro assessment of porcine neutrophil activation responses to chemoattractants: flow cytometric evidence for the selective absence of formyl peptide receptors.

Interest in the role that activated granulocytes play in C5a‐induced myocardial ischemia prompted us to investigate and compare activation responses of pig and human neutrophils. The responses of Hypaque‐Ficoll purified porcine (P‐PMN) and human neutrophils (H‐PMN) to stimulation with N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP), C5a, phorbol myristate acetate (PMA), and calcium ionophore A23187 (A23187) were compared by flow cytometrically measured changes in the cells forward (FWD‐SC) (a measure of shape/volume change) and right angle (90°‐SC) light scatter (a measure of secretion), and in the distribution of the membrane potential sensitive fluorescent probe di‐O‐C(5) (3). FMLP, C5a, and Zymosan‐activated serum (ZAS) stimulated chemotaxis and FMLP vs. PMA‐stimulated adherence to plastic were also compared. Unstimulated P‐PMN had lower FWD‐SC and 90°‐SC than H‐PMN (39.4 ± 1.4 vs. 48.4 ± 2.0 P < 0.05, and 32.7 ± 2.7 vs. 52.4 ± 1.5 units, P < 0.005, for FWD‐SC and 90°‐SC of P‐PMN vs. H‐PMN, respectively). P‐PMN selectively failed to increase their FWD‐SC upon stimulation with FMLP (0.0 ± 0.5% vs. 26.1 ± 6.8%, P‐PMN vs. H‐PMN), or decrease their 90°‐SC when treated with cytochalasin B + FMLP (secretion) (2.4 ± 0.1% vs. ‐35.8 ± 4.6% change in 90°‐SC, P‐PMN vs. H‐PMN), while responding comparably to C5a, PMA, and A23187. P‐PMN failed to depolarize in response to FMLP but responded similarly to H‐PMN when activated by C5a, A23187, and PMA. P‐PMNs chemotactic response to FMLP was selectively absent since the cells responded well to purified pig C5a. FMLP stimulated significant increases in H‐PMN adherence to bovine serum albumin‐coated plastic (44.1 ± 6.7% vs. 12.6 ± 3.7%, FMLP vs. buffer, P < 0.025), but failed to increase adherence of P‐PMN above baseline 0.68 ± 0.20% vs. 2.12 ± 1.90%, FMLP vs. buffer, P > 0.05. PMA (100 ng/ml) stimulated comparable increases in adherence in both PMN types (48.6 ± 5.2% vs. 58.7 ± 4.9%, P‐PMN vs. H‐PMN, P < 0.025). Binding studies using the fluoresceinated N‐formyl peptide f‐met‐leu‐phe‐lysine‐fluorescein‐isothiocyanate (FMLPL‐FITC) in the absence and presence of excess non‐fluoresceinated FMLPL indicated that P‐PMN lack specific binding sites for the N‐formyl peptides. Intracoronary (LAD) infusion of FMLP in the instrumented intact pig produced no change in neutrophil extraction, LAD regional blood flow, or myocardial contractility while infusion of purified porcine C5a induced a rapid and marked increase in myocardial neutrophil extraction, a decrease in LAD coronary blood flow, and diminished contractility. It is concluded that P‐PMN and H‐PMN respond comparably to C5a, PMA, and A23187, but P‐PMN are selectively unresponsive to activation by FMLP both in vitro and in vivo due to the absence of FMLP binding.

Role of thromboxane A2 in the cardiovascular response to intracoronary C5a.

Intracoronary administration of complement component C5a induces transient decreases in coronary blood flow and regional left ventricular segment shortening, associated with intramyocardial granulocyte trapping. We evaluated the influence of a cyclooxygenase inhibitor (acetylsalicylic acid, n = 8) or a thromboxane A2/prostaglandin H2 receptor antagonist (SQ29548, n = 6) on these C5a-induced cardiovascular responses. Open-chest anesthetized pigs were instrumented to monitor heart rate, arterial blood pressure, left anterior descending coronary blood flow, regional left ventricular segment shortening, and dP/dt. Oxygen content, lactate concentration, leukocyte count, and thromboxane B2, the stable metabolite of thromboxane A2, were measured in arterial and regional coronary venous blood. Repetitive injections of intracoronary C5a (500 ng) given 60 minutes apart showed no tachyphylaxis of the hemodynamic response. However, tachyphylaxis was seen in coronary blood flow changes when injections were spaced 30 minutes apart. An increase in myocardial oxygen extraction and lactate production was observed after intracoronary C5a. Administration of acetylsalicylic acid (50 mg/kg i.v.) attenuated C5a-induced decreases in coronary blood flow (-8 +/- vs. -3 +/- 1 ml/min) and regional left ventricular segmental shortening (-10 +/- 3% vs. -2 +/- 1%) and blocked the maximal increase in coronary venous thromboxane B2 (2.0 +/- 0.1 vs. 0.2 +/- 0.1 pmol/ml plasma). Furthermore, SQ29548 (30 micrograms/kg/min) reduced C5a-induced changes in coronary blood flow (-13 +/- 2 vs. -4 +/- 2 ml/min) and segmental shortening (-14 +/- 2% vs. -3 +/- 1%). Neither cyclooxygenase inhibition nor thromboxane A2/prostaglandin H2 antagonism blocked the decrease in coronary venous granulocyte count.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrogen peroxide-induced cardiovascular reflexes. Role of hydroxyl radicals.

Mesenteric ischemia reflexly activates the cardiovascular system. In addition, mesenteric ischemia and reperfusion generate reactive oxygen species. However, the ability of these short-lived reactive oxygen species to generate cardiovascular reflexes is unknown. We therefore investigated cardiovascular reflexes induced by serosal application of hydrogen peroxide (H2O2) to the gallbladder, stomach, or duodenum in anesthetized cats. Serosal application of hydrogen peroxide (44 mumols) to the gallbladder (n = 14) significantly (p less than 0.05) increased mean arterial blood pressure (MAP) by 37 +/- 6 mm Hg, left ventricular dP/dt by 1,893 +/- 416 mm Hg/sec, heart rate by 6 +/- 1 beats per minute, and systemic vascular resistance from 0.34 +/- 0.01 to 0.42 +/- 0.04 peripheral resistance units. The cardiovascular effects were dose-dependent over a range of 0.4 pmol to 132 mumols H2O2. Celiac and superior mesenteric ganglionectomy abolished H2O2-induced cardiovascular effects. Dimethylthiourea (10 mg/kg), a reactive oxygen species scavenger, significantly (p less than 0.05) attenuated 44 mumols H2O2-induced increases in MAP from 36 +/- 3 to 2 +/- 2 mm Hg. Deferoxamine (10 mg/kg) also significantly attenuated 44 mumols H2O2-induced increases in MAP from 40 +/- 7 to 19 +/- 10 mm Hg, but iron-loaded deferoxamine did not. Aspirin (50 mg/kg) did not attenuate H2O2-induced excitation of the cardiovascular system. These data suggest that H2O2 activates abdominal visceral afferents to reflexly stimulate the cardiovascular system by a mechanism involving hydroxyl radicals. Thus, reactive oxygen species could modulate systemic vascular tone by stimulating abdominal visceral afferents during mesenteric ischemia and reperfusion.