PKC activation increases Ca²âº sensitivity of permeabilized lymphatic muscle via myosin light chain 20 phosphorylation-dependent and -independent mechanisms.

The contractile activity of muscle cells lining the walls of collecting lymphatics is responsible for generating and regulating flow within the lymphatic system. Activation of PKC signaling contributes to the regulation of smooth muscle contraction by enhancing sensitivity of the contractile apparatus to Ca(2+). It is currently unknown whether PKC signaling contributes to the regulation of lymphatic muscle contraction. We hypothesized that the activation of PKC signaling would increase the sensitivity of the lymphatic myofilament to Ca(2+). To test this hypothesis, we determined the effects of PKC activation with phorbol esters [PMA or phorbol dibutyrate (PDBu)] on the contractile behavior of α-toxin-permeabilized rat mesenteric and cervical lymphatics or the thoracic duct. The addition of PMA or PDBu induced a significant increase in the contractile force of submaximally activated α-toxin-permeabilized lymphatic muscle independent of a change in intracellular Ca(2+) concentration, and the Ca(2+)-force relationship of lymphatic muscle was significantly left shifted, indicating greater myofilament Ca(2+) sensitivity. Phorbol esters increased the maximal rate of force development, whereas the rate of relaxation was reduced. Western blot and immunohistochemistry data indicated that the initial rapid increase in tension development after stimulation by PDBu was associated with myosin light chain (MLC)20 phosphorylation; however, the later, steady-state Ca(2+) sensitization of permeabilized lymphatic muscle was not associated with increased phosphorylation of MLC20 at Ser(19), 17-kDa C-kinase-potentiated protein phosphatase-1 inhibitor at Thr(38), or caldesmon at Ser(789). Thus, these data indicate that PKC-dependent Ca(2+) sensitization of lymphatic muscle may involve MLC20 phosphorylation-dependent and -independent mechanism(s).

Divergent effects of aging and sex on vasoconstriction to endothelin in coronary arterioles.

OBJECTIVE: The risk for cardiovascular disease increases with advancing age; however, the chronological development of heart disease differs in males and females. The purpose of this study was to determine whether age-induced alterations in responses of coronary arterioles to the endogenous vasoconstrictor, endothelin, are sex-specific. METHODS: Coronary arterioles were isolated from young and old male and female rats to assess vasoconstrictor responses to endothelin (ET), and ETa and ETb receptor inhibitors were used to assess receptor-specific signaling. RESULTS: In intact arterioles from males, ET-induced vasoconstriction was reduced with age, whereas age increased vasoconstrictor responses to ET in intact arterioles from female rats. In intact arterioles from both sexes, blockade of either ETa or ETb eliminated age-related differences in responses to ET; however, denudation of arterioles from both sexes revealed age-related differences in ETa-mediated vasoconstriction. In arterioles from male rats, ETa receptor protein decreased, whereas ETb receptor protein increased with age. In coronary arterioles from females, neither ETa nor ETb receptor protein changed with age, suggesting age-related changes in ET signaling occur downstream of ET receptors. CONCLUSIONS: Thus, aging-induced alterations in responsiveness of the coronary resistance vasculature to endothelin are sex-specific, possibly contributing to sexual dimorphism in the risk of cardiovascular disease with advancing age.

Calcium sensitivity and cooperativity of permeabilized rat mesenteric lymphatics.

Lymphatic muscle contraction is critical for the centripetal movement of lymph that regulates fluid balance, protein homeostasis, lipid absorption, and immune function. We have demonstrated that lymphatic muscle has both smooth and striated muscle contractile elements; however, the basic contractile properties of this tissue remain poorly defined. We hypothesized that contractile characteristics of lymphatic myofilaments would be different from vascular smooth muscle myofilaments. To test this hypothesis, -log[Ca(2+)] (pCa)-tension relationship was determined for alpha-toxin permeabilized mesenteric lymphatics, arteries, and veins. The Ca(2+) sensitivity (pCa(50)) of mesenteric lymphatics was significantly lower compared with arteries (6.16 +/- 0.05 vs. 6.44 +/- 0.02; P < 0.05), whereas there was no difference in pCa(50) between lymphatics and veins (6.16 +/- 0.05 vs. 6.00 +/- 0.10; not significant). The Hill coefficient for alpha-toxin-permeabilized lymphatics was not significantly different from arteries but was significantly greater than that of the veins (1.98 +/- 0.19 vs. 1.21 +/- 0.18; P < 0.05). In addition, the maximal tension and pCa(50) values were significantly greater in alpha-toxin-permeabilized lymphatics compared with beta-escin-permeabilized lymphatics (0.27 +/- 0.03 vs. 0.15 +/- 0.01 and 6.16 +/- 0.05 vs. 5.86 +/- 0.06 mN/mm, respectively; P < 0.05), whereas the Hill coefficient was significantly greater in beta-escin-permeabilized lymphatics. Western blot analyses revealed that CPI-17 levels were significantly decreased by about 50% in beta-escin-permeabilized lymphatics, compared with controls, whereas no change in the level of calmodulin was detected. Our data constitute the first description of the pCa-tension relationship in permeabilized lymphatic muscle. It suggests that differences in myofilament Ca(2+) sensitivity and cooperativity among lymphatic muscle and vascular smooth muscles contribute to the functional differences that exist between these tissues.

Comparison of cardiorespiratory responses of moderately trained men and women using two different treadmill protocols.

In practice, the Bruce protocol is the most commonly used treadmill protocol to assess maximal oxygen consumption (V(.-)O2max). It has been suggested that a running protocol (e.g., Astrand) may elicit a comparatively higher V(.-)O2max and different cardiorespiratory responses when applied to moderately trained runners. Thus, the purpose of this study was to compare V(.-)O2max and other cardiorespiratory responses as elicited by the standard Bruce and a modified Astrand treadmill protocol in moderately trained runners. Fifteen women (age = 21 years, height = 171.5 cm, weight = 63 kg, and body fat = 18%) and 15 men (age = 26 years, height = 177 cm, weight = 72 kg, and body fat = 9%) who were moderately trained runners completed a standard Bruce and modified Astrand protocol (random order), separated by approximately 7 days. Heart rate, Borg ratings of perceived exertion, blood pressure, and pulmonary gas exchange variables were measured during the exercise tests using standard laboratory procedures. This study revealed V(.-)O2max values between the Bruce protocol (51.3 +/- 11.6 ml x kg(-1) x min(-1)) and modified Astrand (51.5 +/- 10.9 ml x kg(-1) x min(-1)) were not significantly different in either the men or the women. However, the Bruce protocol elicited significantly higher maximum treadmill time in men and maximum respiratory exchange ratio (RERmax) and maximum minute ventilation (VEmax) values in both genders. Conversely, the modified Astrand elicited a higher HRmax. These data suggest that V(.-)O2max in both moderately trained men and women runners is independent of treadmill protocol despite differences in HRmax, RERmax, and VEmax.

Effects of aging on microvascular oxygen pressures in rat skeletal muscle.

Aging alters skeletal muscle vascular geometry and control such that the dynamics of muscular blood flow (Q) and O2 delivery (Q(O2)) may be impaired across the rest-exercise transition. If, at the onset of muscle contractions, Q dynamics are slowed disproportionately to those of muscle O2 uptake (V(O2), microvascular PO2 (PO2m) would be reduced and blood-tissue O2 transfer compromised. This investigation determined the effects of aging on PO2m (a direct reflection of the Q(O2)-to-V(O2) ratio), at rest and across the rest-contractions transition in the spinotrapezius of young (approximately 6 months, n = 9) and old (>24 months, n = 10) male Fisher 344/Brown Norway hybrid rats. Phosphorescence quenching techniques were used to quantify PO2m, and test the hypothesis that, across the rest-contractions (twitch, 1 Hz; 4-6 V, 240 s) transition, aging would transiently reduce the Q(O2)-to-V(O2) ratio causing a biphasic profile in which PO2m fell below steady-state contracting values. Old rats had a lower pre-contraction baseline PO2m than young (27.1+/-1.9 versus 33.8+/-1.6 mmHg, P<0.05, respectively). In addition, in old rats PO2m demonstrated a pronounced difference between the absolute nadir and end-contracting values (2.5+/-0.9 mmHg), which was absent in young rats. In conclusion, unlike their young counterparts, old rats exhibited a transiently reduced PO2m across the rest-contractions transition that may impair blood-tissue O2 exchange and elevate the O2 deficit, thereby contributing to premature fatigue.