Ageing diminishes endothelium-dependent vasodilatation and tetrahydrobiopterin content in rat skeletal muscle arterioles

Abstract

Ageing reduces endothelium-dependent vasodilatation through an endothelial nitric oxide synthase (NOS) signalling pathway. The purpose of this study was to determine whether arginase activity diminishes endothelium-dependent vasodilatation in skeletal muscle arterioles from old rats, and whether NOS substrate (L-arginine) and cofactor (tetrahydrobiopterin; BH(4)) concentrations are reduced. First-order arterioles were isolated from the soleus muscle of young (6 months old) and old (24 months old) male Fischer 344 rats. In vitro changes in luminal diameter in response to stepwise increases in flow were determined in the presence of the NOS inhibitor N(G)-nitro-L-arginine methyl ester (l-NAME, 10(-5) mol l(-1)), the arginase inhibitor N(omega)-hydroxy-nor-L-arginine (NOHA, 5 x 10(-4) mol l(-1)), exogenous L-arginine (3 x 10(-3) mol l(-1)) or the precursor for BH(4) synthesis sepiapterin (1 micromol l(-1)). Arteriolar L-arginine and BH(4) content were determined via HPLC. Ageing decreased flow-mediated vasodilatation by 52%, and this difference was abolished with NOS inhibition. Neither inhibition of arginase activity nor addition of exogenous L-arginine had any effect on flow-mediated vasodilatation; arteriolar l-arginine content was also not different between age groups. BH(4) content was lower in arterioles from old rats (94 +/- 8 fmol (mg tissue)(-1)) relative to controls (234 +/- 21 fmol (mg tissue)(-1)), and sepiapterin elevated flow-mediated vasodilatation in arterioles from old rats. These results demonstrate that the impairment of endothelium-dependent vasodilatation induced by old age is due to an altered nitric oxide signalling mechanism in skeletal muscle arterioles, but is not the result of increased arginase activity and limited L-arginine substrate. Rather, the age-related deficit in flow-mediated vasodilatation appears to be the result, in part, of limited BH(4) bioavailability.

Figure 1. Effects of ageing and l-NAME on flow-induced vasodilatation in soleus muscle arterioles from young and old rats

*Indicates vasodilatation in response to flow was lower in soleus muscle arterioles from aged rats (P < 0.05). l-NAME reduced flow-induced vasodilatation in arterioles from young and old rats (P < 0.05) and eliminated differences in responsiveness to flow between the young and old. Values are means ±s.e.m. (n = 12–14 per group).

Figure 2. Effects of ageing and arginase inhibition (via N^ω-hydroxy-nor-l-arginine; NOHA) on flow-induced vasodilatation in soleus muscle arterioles from young and old rats

*Indicates vasodilatation in response to flow was lower in soleus muscle arterioles from old rats (P < 0.05). NOHA did not affect the flow-induced vasodilatation in arterioles from either young or old rats. Values are means ±s.e.m. (n = 9 per group).

Figure 3. Effects of ageing and exogenous l-arginine (3 × 10⁻³ mol l⁻¹) on flow-induced vasodilatation in soleus muscle arterioles from young and old rats

*Indicates vasodilatation in response to flow was lower in soleus muscle arterioles from old rats (P < 0.05). l-arginine did not affect the flow-induced vasodilatation in arterioles from either young or old rats. Values are means ±s.e.m. (n = 9–11 per group).

Figure 4. Effects of ageing and sepiapterin (1 μmol l⁻¹) on flow-induced vasodilatation in soleus muscle arterioles from young and old rats

*Indicates vasodilatation in response to flow was different between conditions (P < 0.05). Values are means ±s.e.m. (n = 13 per group).

Figure 5. l-Arginine concentration in arterioles from the soleus muscle of young and old rats

Ageing had no effect on arteriolar l-arginine content (P > 0.05). n = 5 per group.

Figure 6. Tetrahydrobiopterin (BH₄) concentration in arterioles from the soleus muscle of young and old rats

*Indicates significant difference in BH₄ content to that of arterioles from young rats (P < 0.05). n = 5 per group.

Figure 7. The proposed effects of ageing on BH₄ bioavailability

Vascular BH₄ content may be limited by reduced synthesis through the de novo or the salvage pathways. Further, because BH₄ is susceptible to oxidative degradation, elevations in the production of reactive oxygen species associated with old age could directly decrease vascular BH₄ concentration. Peroxynitrite (ONOO·), a product of nitric oxide (NO) and superoxide (O₂·) generated through NADPH oxidases and other sources, including xanthine oxidase, cytochrome P-450 enzymes and mitochondria, could further increase catabolism of BH₄. With decreased levels of BH₄, eNOS becomes uncoupled and leads to generation of O₂· rather than NO. Thus, the uncoupling of eNOS decreases the NO available to mediate smooth muscle cell relaxation and increases O₂· generation, which could further depress BH₄ bioavailability.