Introduction
As we've shown previously, myofilament calcium sensitivity is upregulated in fetal compared to adult cerebral arteries, particularly in response to G-protein receptor agonists. Myogenic reactivity, however, has been suggested to be depressed in immature compared to mature arteries. The present experiments explore the hypothesis that myogenic reactivity is depressed in immature arteries due to attenuation of stretch-induced calcium influx.
Methods
Posterior communicating arteries from term fetal and non-pregnant adult sheep were mounted in vitro for measurement of contractility and exposed to graded stretch between 1.3 and 2.3 times unstressed diameter. In one segment from each animal, the time course of the effects of rapid stretch on cytosolic calcium were determined via Fura-2 photometry. In the remaining segments, the effects of rapid stretch on myosin light chain phosphorylation were determined using urea gels and immunoblotting for myosin light chain.
Results
The magnitude of maximum myogenic tone as a percentage of the maximum contractile response to potassium was similar in adult (26±5%) and fetal (24±7%) arteries, although peak myogenic tone occurred at a lower stretch ratio in fetal (2.1) than adult (2.3) arteries. Graded stretch increased cytosolic calcium significantly more in adult (ΔR=0.292) than in fetal (ΔR=0.108) arteries. Unexpectedly, stretch increased %myosin phosphorylation to similar peak values in fetal (45±8%) and adult (41±7%) arteries. Basal phosphorylation levels were also similar in fetal (14±3%) and adult (11±3%) arteries. The stretch ratios at which peak phosphorylation was observed were far less in both the fetus (1.9) and adult (1.9) than were required to produce maximum myogenic tone. Plots of %myosin phosphorylation against stretch yielded a sigmoidal relation in adult arteries, indicating stretch-induced thin filament activation. This relation was absent in fetal arteries.
Discussion
The present results refute the idea that myogenic tone is depressed in immature cerebral arteries, when tone is normalized relative to maximum potassium-induced contractile capacity. Stretch-induced increases in cytosolic calcium were indeed significantly less in fetal than adult arteries, but the extent of stretch-induced myosin phosphorylation was similar in both age groups. This suggests either that the ability of calcium to activate myosin light chain kinase is greater in fetal than adult arteries and/or that myosin phosphatase activity is attenuated in immature relative to mature arteries, particularly during graded stretch. Our finding of a direct relation between %myosin phosphorylation and myogenic tone also suggests a possible mechanism coupling stretch to thin-filament activation. Because this relation was observed in adult but not fetal arteries, it may help explain why myogenic length-tension relations operate over a greater range in mature arteries, and why peak myogenic tone develops at stretch ratios greater than required to produced peak myosin phosphorylation. Whereas the mechanisms coupling stretch to thin filament activation clearly merit further study, it is clear that fetal artery responses to stretch are mediated by a different mix of mechanisms that those governing mature arteries. Regardless, fetal arteries exhibit a well-developed myogenic responses to stretch that no doubt contribute to cerebral autoregulation in the fetus and neonate.
Footnotes
Acknowledgements
Supported by NIH HD31226 and HL54120.