Maximal stimulation-induced in situ myosin light chain kinase activity is upregulated in fetal compared with adult ovine carotid arteries

Abstract

Postnatal decreases in vascular reactivity involve decreases in the thick filament component of myofilament calcium sensitivity, which is measured as the relationship between cytosolic calcium concentration and myosin light chain (MLC20) phosphorylation. The present study tests the hypothesis that downregulation of thick filament reactivity is due to downregulation of myosin light chain kinase (MLCK) activity in adult compared with fetal arteries. Total MLCK activity, calculated as %MLC20 phosphorylated per second in intact arteries during optimal inhibition of myosin light chain phosphatase activity, was significantly less in adult (6.56+/-0.29%) than in fetal preparations (7.39+/-0.53%). In situ MLC20 concentrations (microM) in adult (198+/-28) and fetal arteries (236+/-44) did not differ significantly. In situ MLCK concentrations (microM), however, were significantly greater in adult (8.21+/-0.59) than in fetal arteries (1.83+/-0.13). In situ MLCK activities (ng MLC20 phosphorylated.s(-1).ng MLCK(-1)) were significantly less in adult (0.26+/-0.01) than in fetal arteries (1.52+/-0.11). In contrast, MLCK activities in adult (15.8+/-1.5) and fetal artery homogenates (17.3+/-1.3) were not significantly different. When in situ fractional activation was calculated, adult values (1.72+/-0.17%) were significantly less than fetal values (9.08+/-0.83%). Together, these results indicate that decreased thick filament reactivity in adult compared with fetal ovine carotid arteries is due at least in part to greater MLCK activity in fetal arteries, which in turn cannot be explained by differences in MLCK, MLC20, or calmodulin concentrations. Instead, this difference appears to involve age-related differences in fractional activation of the MLCK enzyme.

Fig. 1.

In situ concentrations of myosin light chain kinase (MLCK) in ovine carotid arteries. Supernatants from artery homogenates containing 20 μg of total protein were loaded on 8% SDS-PAGE gels along with varying masses of a calibrated standard for MLCK in adjacent lanes. The integrated density values of MLCK were converted to their respective masses using a standard curve and then normalized to intracellular volume using previously published ratios of intracellular water to total cellular protein. In all cases, the band densities for unknown samples were within the range defined by the standards. For the standard bands shown (top), the band density given by 5 μg of MLCK was not visible by eye but was detectable by the gel analysis software (Chemi-Imager; Alpha Innotech). Values indicate the ratio of MLCK mass to intracellular water, expressed as in situ concentration (bottom). Error bars indicate SE for 7 fetal and 7 adult animals. The average value was significantly less in fetal than in adult arteries at the P < 0.05 level.

Fig. 2.

In situ concentrations of myosin light chain (MLC₂₀) and calmodulin in ovine carotid arteries. Artery homogenates were loaded on 15% SDS-PAGE gels along with varying masses of calibrated standards for either MLC₂₀ (left) or calmodulin (right) in adjacent lanes. The integrated density values of each protein were converted to their respective masses by using their respective standard curves and then normalized to intracellular volume using previously published ratios of intracellular water to total cellular protein. Values indicate in situ concentrations of calmodulin and MLC₂₀ for both fetal and adult arteries. Error bars indicate SE for the number of animals indicated in parentheses. There was a significant difference between the fetal and adult values at the P < 0.05 level for calmodulin.

Fig. 3.

Length-tension relations in ovine carotid arteries. Carotid artery segments of fetal and adult sheep were wire mounted and stretched to regular multiples of unstressed baseline diameter (D/D₀) ranging from 1.0 to 3.0. At each stretch ratio, contractile responses to 122 mM K⁺ were recorded and then normalized relative to maximum response. Error bars indicate SE for a total of 10 artery segments from 5 fetuses and 12 artery segments from 6 adult sheep.

Fig. 4.

Concentration- and time-dependent effects of ML-7 on MLCK activity. Concentration-dependent effects of ML-7 on MLCK activity (top) were assessed by treatment with 0, 10, 30, 100, or 300 μM ML-7 for 30 min, followed by incubation with 10 μl/ml phosphatase inhibitor cocktail (PIC) for an hour. The segments were then instantly frozen after 9 s of contraction with 122 mM K⁺ and analyzed for MLC₂₀ phosphorylation. Time courses of MLC₂₀ phosphorylation were then determined in the presence and absence of 100 μM ML-7 (bottom) by freezing at 0, 3, 6, and 9 s after contraction with 122 mM K⁺. A total of 65 segments each from 5 fetuses and 5 adult arteries were used for this study. Values indicate averages ± SE for the number of fetal and adult sheep indicated in parentheses. *P < 0.05, significant difference from initial control value within each age group.

Fig. 5.

MLCK velocity in intact ovine carotid arteries. Fetal and adult artery segments were wire mounted and positioned in a custom-made rapid freeze apparatus, activated via electrical field stimulation, and frozen at exactly 0, 1, 2, and 3 s using a computer-controlled apparatus. The segments were then analyzed for MLC₂₀ phosphorylation. The initial rate of change of MLC₂₀ phosphorylation was taken as a measure of MLCK velocity (top). To correct for age-related differences in MLC₂₀ and MLCK abundances, the %MLC₂₀ phosphorylation ratios were multiplied by the corresponding mass ratios of MLC₂₀/MLCK abundance to obtain units of ng MLC₂₀ phosphorylated/ng MLCK (bottom). The initial slopes (V) are given for each age group. A total of 20 artery segments from 5 fetuses and 20 artery segments from 5 adults were used for this protocol. Values indicate averages ± SE. *P < 0.05, significant difference between corresponding fetal and adult value. Error bars for adult values at bottom are smaller than the size of the symbols.

Fig. 6.

Age-related differences in the apparent specific activity and fractional activation of MLCK. Apparent specific activity was calculated as the maximum velocity divided by MLCK content (left). Fractional activation of the in situ enzyme was calculated as the ratio of maximal velocity measured in intact arteries over the maximal velocity measured in the homogenates (right). Shown are averages for 6 animals from each group for specific activity measurements and 5 animals from each group for fractional activation. Error bars indicate SE for the number of animals indicated in parentheses. There was a significant difference between the fetal and adult values at the P < 0.05 level for fractional activation.