Role of nitrite in regulation of fetal cephalic circulation in sheep

Abstract

Nitrite has been postulated to provide a reservoir for conversion to nitric oxide (NO), especially in tissues with reduced oxygen levels as in the fetus. Nitrite would thus provide local vasodilatation and restore a balance between oxygen supply and need, a putative mechanism of importance especially in the brain. The current experiments test the hypothesis that exogenous nitrite acts as a vasodilator in the cephalic vasculature of the intact, near term fetal sheep. Fetuses were first instrumented to measure arterial blood pressure and carotid artery blood flow and then studied 4-5 days later while in utero without anaesthesia. Initially l-nitro-arginine (LNNA) was given to block endogenous NO production. Carotid resistance to flow increased 2-fold from 0.54 ± 0.01 (SEM) to 1.20 ± 0.08 mmHg min ml(-1) (in 13 fetuses, P < 0.001), indicating NO tonically reduces cerebral vascular tone. Sodium nitrite (or saline as control) was then infused in increasing step-doses from 0.01 to 33 μm in half-log increments over a period of 2 h. Carotid artery pressure, blood flow and vascular resistance did not change compared to fetuses receiving saline, even at plasma nitrite concentrations two orders of magnitude above the physiological range. The results indicate that while cephalic vascular tone is controlled by endogenous nitric oxide synthase activity, exogenously administered nitrite is not a vasodilator at physiological concentrations in the vasculature served by the carotid artery of fetal sheep.

Figure 1. Protocol of nitrite infusion into carotid artery of fetal sheep

LNNA was given intravenously to the fetus (−120 min) and ewe (−60 min) and nitrite or saline infusions into a fetal carotid artery were begun at time 0. Nitrite infusion increased systemic fetal blood nitrite concentrations from ∼10 nm (infusion ‘a’) to 33,333 nm (infusion ‘h’) in half-log incremental steps every 15 min. Physiological concentrations were estimated to occur during steps ‘c’ to ‘e’. Concentrations of nitrite in the carotid artery were calculated based on a unilateral carotid flow of 50 ml min⁻¹.

Figure 2. Arterial blood gas responses to infusion of nitrite or saline in fetal sheep

Results are shown for fetuses that received LNNA + saline (squares, n = 6), LNNA + nitrite (circles, n = 7), and saline + nitrite (triangles, n = 4). Open symbols denote values significantly different from baseline values for that group (1-way ANOVA with Bonferroni post hoc analysis). Differences between study groups are not significant (2-way ANOVA). Shaded area shows time period of increasing nitrite dosage.

Figure 3. Arterial nitrite concentrations

A, brachial artery nitrite concentrations in response to LNNA infusion. B, nitrite concentrations during nitrite infusion in blood sampled from the brachial artery. C, calculated nitrite concentrations in the carotid artery downstream of the site of nitrite infusion, with values based on carotid blood flow, rate of nitrite infusion, and concentration of nitrite measured in the brachial arterial blood. Note that nitrite levels range from less than initial baseline to 100-fold higher than normal at the maximal infusion rate. Squares represent fetuses that received LNNA + saline (n = 6), circles represent fetuses that received LNNA + nitrite (n = 7), and triangles represent the fetuses that received saline + nitrite (n = 4). In A, open symbols denote time points that were significantly different from the −135 min time point. In B and C, open symbols denote time points significantly different from the 0 min time point (1-way ANOVA with Bonferroni post hoc analysis). (⁺P < 0.05 for comparison between LNNA + nitrite and LNNA + saline groups. *P < 0.05 for comparison between the saline + nitrite and LNNA + nitrite groups (2-way ANOVA with Bonferroni post hoc analysis).

Figure 4. Changes in mean arterial blood pressure, heart rate, carotid blood flow and carotid resistance in response to LNNA bolus followed by infusion of saline or nitrite in stepwise increasing concentrations during the shaded area

Squares represent fetuses receiving LNNA + saline (n = 6), circles represent fetuses receiving LNNA + nitrite (n = 7), and triangles represent fetuses receiving saline + nitrite (n = 4). Open symbols represent time points that are significantly different from the pre-nitrite infusion values measured at time 0 (1-way ANOVA, Bonferroni post hoc test). LNNA bolus to the fetuses resulted in significant increases in mean arterial blood pressure and carotid vascular resistance to flow, and decreases in heart rate (beats min^–1) and carotid blood flow (1-way ANOVA). There were no significant differences between the LNNA + nitrite and LNNA + saline groups. *P < 0.05 for comparison between the LNNA + nitrite and the saline + nitrite groups (2-way ANOVA with Bonferroni post hoc test).

Figure 5. Effect of intra-carotid infusion of an NO donor on carotid vascular resistance

Three of the fetuses that received nitrite but no LNNA were given a bolus of PROLI-NO into the carotid artery at time 0, resulting in a significant decrease in carotid vascular resistance and demonstrating the capacity of the vasculature to respond to NO (P < 0.0001, 1-way ANOVA).

Figure 6. Arterial blood pressure, carotid flow and carotid resistance in response to initial NOS blockade with intravenous LNNA infusion (squares) and a repeat exposure to intravenous LNNA 2 days later (circles)

Open symbols represent time points that were significantly different from the baseline (−135 min) value (1-way ANOVA with Bonferroni post hoc analysis). The significant increases in blood pressure, decreases in carotid flow and increases in arterial resistance seen after the first exposure to LNNA were not seen with re-exposure to LNNA 2 days later. *Significant difference between 1st exposure and 2nd exposure (2-way ANOVA with Bonferroni post hoc test).

Figure 7. Plasma CGRP concentrations prior to the initial infusion of LNNA (Pre-LNNA) and the second infusion of LNNA 2 days later (Post-LNNA)

Plasma CGRP concentrations increased significantly in plasma collected 2 days after exposure to LNNA (paired t test).