In vascular smooth muscle, elevation of agonist-induced intracellular Ca2+ concentration ([Ca2+]i) occurs via both Ca2+ release from intracellular stores and Ca2+influx across the plasma membrane. In the cerebral vasculature of the fetus and adult the relative roles of these mechanisms have not been defined. To test the hypothesis that plasma membrane L-type and receptor-operated Ca2+ channels play a key role in NE-induced vasoconstriction via alterations in plasma membrane Ca2+ flux and that this may change with developmental age, we performed the following study. In main branch middle cerebral arteries (MCA) from near-term fetal (∼140 days) and nonpregnant adult sheep, we quantified NE-induced responses of vascular tension and [Ca2+]i(by use of fura 2) under standard conditions in response to several Ca2+ channel blockers and in response to zero extracellular Ca2+. In fetal and adult MCA, maximal NE-induced tensions (g) were 0.91 ± 0.12 ( n = 10) and 1.61 ± 0.13 ( n = 12), respectively. The pD2 values for NE-induced tension were both 6.0 ± 0.1, whereas the fetal and adult maximum responses (%Kmax) were 107 ± 16 and 119 ± 7, respectively. The fetal and adult pD2 values for NE-induced increase of [Ca2+]iwere 6.2 ± 0.1 and 6.4 ± 0.1, respectively, whereas maximum [Ca2+]iresponses were 81 ± 9 and 103 ± 15% of Kmax, respectively. After 10−5 M NE-induced contraction, nifedipine resulted in dose-dependent decrease in vessel tone and [Ca2+]iwith pIC50 values for fetal and adult tensions of 7.3 ± 0.1 and 6.6 ± 0.1, respectively ( P < 0.01; n = 4 each), whereas pIC50 for [Ca2+]iresponses were 7.2 ± 0.1 and 6.9 ± 0.1, respectively. The pIC50 values for tension for diltiazem and verapamil were somewhat lower but showed a similar relationship. The receptor-operated Ca2+ channel blocker 2-nitro-4 carboxyphenyl- N,N-diphenyl carbamate showed little effect on NE-induced vessel contractility or [Ca2+]i. In the absence of extracellular Ca2+ for 2 min, 10−5 M NE resulted in markedly attenuated responses of adult MCA tension and [Ca2+]ito 39 ± 7 and 73 ± 8% of control values ( n = 4). For fetal MCA, exposure to extracellular Ca2+concentration resulted in essentially no contractile or [Ca2+]iresponse ( n = 4). Similar blunting of NE-induced tension and [Ca2+]iwas seen in response to 10−3M lanthanum ion. These findings provide evidence to suggest that especially in fetal, but also in adult, ovine MCA, Ca2+ flux via L-type calcium channels plays a key role in NE-induced contraction. In contrast, Ca2+ flux via receptor-operated Ca2+ channels is of less importance. This developmental difference in the role of cerebrovascular plasma membrane Ca2+ channels may be an important association with increased Ca2+sensitivity of the fetal vessels.
To test the hypothesis that sarcoplasmic reticulum (SR) Ca(2+) stores play a key role in norepinephrine (NE)-induced contraction of fetal and adult cerebral arteries and that Ca(2+) stores change with development, we performed the following study. In main branch middle cerebral arteries (MCA) from near-term fetal ( approximately 140 days) and nonpregnant adult sheep, we measured NE-induced contraction and intracellular Ca(2+) concentration ([Ca(2+)](i)) in the absence and presence of different blockers. In adult MCA, after thapsigargin (10(-6) M), the NE-induced responses of tension and [Ca(2+)](i) were 37 +/- 5 and 47 +/- 7%, respectively, of control values (P < 0.01 for each). In the fetal artery, in contrast, this treatment resulted in no significant changes from control. When this was repeated in the absence of extracellular Ca(2+), adult MCA increases in tension and [Ca(2+)](i) were 32 +/- 5 and 13 +/- 3%, respectively, of control. Fetal cerebral arteries, however, showed essentially no response. Ryanodine (RYN, 3 x 10(-6) to 10(-5) M) resulted in increases in tension and [Ca(2+)](i) in both fetal and adult MCA similar to that seen with NE. For both adult and fetal MCA, the increased tension and [Ca(2+)](i) responses to RYN were essentially eliminated in the presence of zero extracellular Ca(2+). These findings provide evidence that in fetal MCA, in contrast to those in the adult, SR Ca(2+) stores are of less importance in NE-induced contraction, with such contraction being almost wholly dependent on Ca(2+) flux via plasma membrane L-type Ca(2+) channels. In addition, they suggest that in both adult and fetal MCA, the RYN receptor is coupled to the plasma membrane Ca(2+)-activated K(+) channel and/or L-type Ca(2+) channel.
Studies in vitro as well as in vivo in rodents have suggested that amino acids (AA) not only serve as substrates for protein synthesis, but also as nutrient signals to enhance mRNA translation and protein synthesis in skeletal muscle. However, the physiological relevance of these findings to normal humans is uncertain. To examine whether AA regulate the protein synthetic apparatus in human skeletal muscle, we infused an AA mixture (10% Travesol) systemically into 10 young healthy male volunteers for 6 h. Forearm muscle protein synthesis and degradation (phenylalanine tracer method) and the phosphorylation of protein kinase B (or Akt), eukaryotic initiation factor 4E-binding protein 1, and ribosomal protein S6 kinase (p70(S6K)) in vastus lateralis muscle were measured before and after AA infusion. We also examined whether AA affect urinary nitrogen excretion and whole body protein turnover. Postabsorptively all subjects had negative forearm phenylalanine balances. AA infusion significantly improved the net phenylalanine balance at both 3 h (P < 0.002) and 6 h (P < 0.02). This improvement in phenylalanine balance was solely from increased protein synthesis (P = 0.02 at 3 h and P < 0.003 at 6 h), as protein degradation was not changed. AA also significantly decreased whole body phenylalanine flux (P < 0.004). AA did not activate Akt phosphorylation at Ser(473), but significantly increased the phosphorylation of both eukaryotic initiation factor 4E-binding protein 1 (P < 0.04) and p70(S6K) (P < 0.001). We conclude that AA act directly as nutrient signals to stimulate protein synthesis through Akt-independent activation of the protein synthetic apparatus in human skeletal muscle.
This study tested the hypothesis that protein kinase C (PKC) has dual regulation on norepinephrine (NE)-mediated inositol 1,4, 5-trisphosphate [Ins (1,4,5)P(3)] pathway and vasoconstriction in cerebral arteries from near-term fetal ( approximately 140 gestational days) and adult sheep. Basal PKC activity values (%membrane bound) in fetal and adult cerebral arteries were 38 +/- 4% and 32 +/- 4%, respectively. In vessels of both age groups, the PKC isoforms alpha, beta(I), beta(II), and delta were relatively abundant. In contrast, compared with the adult, cerebral arteries of the fetus had low levels of PKC-epsilon. In response to 10(-4) M phorbol 12,13-dibutyrate (PDBu; PKC agonist), PKC activity in both fetal and adult cerebral arteries increased 40-50%. After NE stimulation, PKC activation with PDBu exerted negative feedback on Ins(1,4,5)P(3) and intracellular Ca(2+) concentration ([Ca(2+)](i)) in arteries of both age groups. In turn, PKC inhibition with staurosporine resulted in augmented NE-induced Ins(1,4,5)P(3) and [Ca(2+)](i) responses in adult, but not fetal, cerebral arteries. In adult tissues, PKC stimulation by PDBu increased vascular tone, but not [Ca(2+)](i). In contrast, in the fetal artery, PKC stimulation was associated with an increase in both tone and [Ca(2+)](i). In the presence of zero extracellular [Ca(2+)], these PDBu-induced responses were absent in the fetal vessel, whereas they remained unchanged in the adult. We conclude that, although basal PKC activity was similar in fetal and adult cerebral arteries, PKC's role in NE-mediated pharmacomechanical coupling differed significantly in the two age groups. In both fetal and adult cerebral arteries, PKC modulation of NE-induced signal transduction responses would appear to play a significant role in the regulation of vascular tone. The mechanisms differ in the two age groups, however, and this probably relates, in part, to the relative lack of PKC-epsilon in fetal vessels.
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