Intrathecal cGMP elicits pressor responses and maintains mean blood pressure during haemorrhage in anaesthetized rats

Abstract

The intracellular second messenger, cyclic guanosine monophosphate (cGMP), a soluble guanylate cyclase (GC) product, is a primary mechanism for the transduction of a nitric oxide (NO)-initiated signal in the central nervous system. NO is produced from L-arginine by neuronal nitric oxide synthase (NOS), which is found in sympathetic preganglionic neurons of the intermediolateral cell column. This suggests the possibility that NO is a modulator of sympathetic nervous activity (SNA) through a cGMP-mediated mechanism. The aim of this study was to determine the effects of intrathecally injected membrane-permeant 8-bromo-cGMP and 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of the soluble form of GC, on arterial pressure in urethane anaesthetized (1.4 g kg(-1) I.P.) rats. The effects of intrathecal cGMP and ODQ on haemodynamic responses to haemorrhage were also investigated. Finally, L-arginine, the NO precursor, was also injected intrathecally, alone and in the presence of ODQ. Baseline mean arterial pressure (MAP) increased significantly after intrathecal 8-Br-cGMP injection (10 microl, 1, 3, 10, 30, 100 microm). A dose-effect relationship (1 microm to 100 microm) was also established (EC(50)=6.03 microm). During continuous haemorrhage, MAP was maintained in animals injected with 8-Br-cGMP, relative to the control group. Although no change in baseline MAP was observed as a result of intrathecal ODQ injection (10 microl, 100 mM), a greater rate of fall in MAP was observed during haemorrhage. Injecting L-arginine (10, 100, 1000 microm, 10 microl) showed a pressor effect that was consistent with the effect of the downstream messenger, cGMP. Furthermore, its pressor effect was blocked by ODQ pre-administration. The results indicate that cGMP increases blood pressure, and thus suggest that cGMP increases SNA. This supports the hypothesis that the sympathoexcitatory effects of spinal delivery of NO are mediated by a cGMP-dependent mechanism.

Figure 1. The dose–response relationship of 8-Br-cGMP (1 μm to 100 μm)

The response is expressed as a change in MAP (mmHg) from baseline as a result of each injection of 8-Br-cGMP (1, 3, 10, 30, 100 μm, 10 μl). The dose–response (Hill) equation has been fitted to the data points ±s.e.m. (▪). EC₅₀= 6.03 μm.

Figure 2. The effect of intrathecal 8-Br-cGMP (2.5 μm, 10 μl) and vehicle (PBS, 10 μl intrathecal) injected prior to haemorrhage (1.5% body weight) on MAP (mmHg ± S.E.M.) measured throughout the removal of blood

MAP during haemorrhage is expressed as a percentage of the initial MAP. *, a significantly different MAP value at x ml blood (100 g body weight)⁻¹ (P < 0.05, Dunnett's post test), compared to the MAP value prior to the onset of haemorrhage; 0 ml blood (100 g body weight)⁻¹. MAP maintenance was observed in cGMP-treated rats till 1.35 ml blood (100 g body weight)⁻¹ had been removed as opposed to maintenance of MAP till 0.90 ml blood (100 g body weight)⁻¹ was removed in the control group. The lines of regression formed by the decrease of MAP over the course of haemorrhage are statistically different (P < 0.01); the control group exhibits a steeper gradient than the group treated with cGMP.

Figure 3. The effect of intrathecal ODQ (100 mm, 10 μl) and vehicle (DMSO, 10 μl intrathecal) injected prior to haemorrhage (1.5% body weight) on MAP (mmHg ±s.e.m.) measured throughout the removal of blood

MAP during haemorrhage is expressed as a percentage of the initial MAP. *, a significantly different MAP value at x ml blood (100 g body weight)⁻¹ (P < 0.05, Dunnett's post test), compared to the MAP value prior to the onset of haemorrhage; 0 ml blood (100 g body weight)⁻¹. MAP maintenance was observed in ODQ-treated rats till 0.60 ml blood (100 g body weight)⁻¹ had been removed as opposed to maintenance of MAP till 0.90 ml ml blood (100 g body weight)⁻¹ had been removed in the control group. The lines of regression formed by the decrease of MAP over the course of haemorrhage are statistically different (P < 0.01); the ODQ-treated group exhibits a steeper gradient than the group treated with cGMP.

Figure 4. The effect of intrathecal 8-Br-cGMP/PBS on HR changes (initial HR versus HR post-haemorrhage)

The cGMP-treated group appears to maintain HR (P > 0.05), whereas in the PBS-treated group, a statistically significant decline in HR is observed (P < 0.05).

Figure 5. The effect of intrathecal l-arginine (l-Arg, middle three bars; 10, 100, 1000 μm, 10 μl injection volume) and vehicle (PBS, leftmost bar; 10 μl volume)

l-Arginine elicits an increase in MAP at all doses (P < 0.05), denoted by an asterisk (*). No changes (ns, P > 0.05), were observed when PBS was given or when l-Arg was injected in ODQ-pretreated rats (rightmost bar; 10 μl of 100 mm ODQ followed by 10 μl of 1 mm l-Arg).