PPHN, caused by perinatal hypoxia or inflammation, is characterized by an increased thromboxane-prostacyclin ratio and pulmonary vasoconstriction. We examined effects of hypoxia on myocyte thromboxane responsiveness. Myocytes from 3rd-6th generation pulmonary arteries of newborn piglets were grown to confluence and synchronized in contractile phenotype by serum deprivation. On the final 3 days of culture, myocytes were exposed to 10% O2 for 3 days; control myocytes from normoxic piglets were cultured in 21% O2. PPHN was induced in newborn piglets by 3-day hypoxic exposure (Fi(O2) 0.10); pulmonary arterial myocytes from these animals were maintained in normoxia. Ca2+ mobilization to thromboxane mimetic U-46619 and ATP was quantified using fura-2 AM. Three-day hypoxic exposure in vitro results in increased basal [Ca2+]i, faster and heightened peak Ca2+ response, and decreased U-46619 EC50. These functional changes persist in myocytes exposed to hypoxia in vivo but cultured in 21% O2. Blockade of Ca2+ entry and store refilling do not alter peak U-46619 Ca2+ responses in hypoxic or normoxic myocytes. Blockade of ryanodine-sensitive or IP3-gated intracellular Ca2+ channels inhibits hypoxic augmentation of peak U-46619 response. Ca2+ response to ryanodine alone is undetectable; ATP-induced Ca2+ mobilization is unaltered by hypoxia, suggesting no independent increase in ryanodine-sensitive or IP3-linked intracellular Ca2+ pool mobilization. We conclude hypoxia has a priming effect on neonatal pulmonary arterial myocytes, resulting in increased resting Ca2+, thromboxane hypersensitivity, and hyperreactivity. We postulate that hypoxia increases agonist-induced TP-R-linked IP3 pathway activation. Myocyte thromboxane hyperresponsiveness persists in culture after removal from the initiating hypoxic stimulus, suggesting altered gene expression.
Neonatal circulatory transition is dependent upon tightly regulated pulmonary circuit relaxation. Persistent pulmonary hypertension of the newborn (PPHN) is characterized by pulmonary arterial myocyte relaxation failure. We examined the effect of short course (72 hour) in vivo normobaric hypoxia in newborn swine on smooth muscle contractile enzyme activity and regulatory phosphoprotein abundance, in tissue homogenates of 2nd to 4th generation pulmonary arteries. Myosin light chain kinase (MLCK) and phosphatase (MLCP) protein contents were unchanged in hypoxic pulmonary arteries compared to controls. MLCP activity increased in normoxic animals from birth to day 3. This was ablated by hypoxia; phosphatase activity, measured as in vitro myosin light chain dephosphorylation, was decreased significantly (P < 0.005) in the hypoxic group. Inhibitory site phosphorylations of MLCP myosin binding subunit at threonines 696 and 850 were similar in both hypoxic and normoxic subjects, suggesting that downregulation of MLCP in hypoxia does not involve this pathway. However, content of regulatory protein CPI-17 (protein kinase C-related phosphatase inhibitor) increased from birth in hypoxic subjects (P < 0.05); active (phosphorylated) CPI-17 protein abundance declined after birth in normals, but increased in hypoxic arteries (P < 0.05). This corresponded with the decrease in phosphatase activity. We speculate that CPI-17 may play a role in myosin phosphatase upregulation during neonatal circulatory transition, and in hypoxic inhibition of pulmonary phosphatase activity in PPHN.
Earlier observations that arachidonic acid inhibited the synthesis of membrane inositol phospholipids in rat submandibular acinar cells prompted the present study on whether the fatty acid may also regulate other key physiological processes in the model. Arachidonate, at concentrations above 10 mumol/L, inhibited up to 97% protein synthesis in acinar cells. The acid also lowered cellular ATP levels to 25% of control values by a ouabain-insensitive mechanism. In endoplasmic reticulum-calcium studies in permeabilized cells, arachidonic acid stimulated the mobilization of up to 73% loaded ER-45Ca2+ to the cytosol, a much greater response than those caused by other calcium translocators, thapsigargin or inositol 1,4,5-trisphosphate. Additionally, arachidonate provoked the release of over 80% of total cell 45Ca2+ to the extracellular space in intact cells and stimulated mucin secretion in the submandibular model. The inhibitory effect of arachidonic acid on protein synthesis was duplicated by carbachol, thapsigargin, and BAPTA/AM, three agents that cause net efflux of ER-Ca2+ by different mechanisms. Furthermore, comparable with the arachidonate effect on ATP, carbachol and thapsigargin also significantly reduced cellular levels of the nucleotide. It is concluded that arachidonic acid acts as a regulator of central synthetic/secretory processes in mucous acinar cells of rat submandibular gland and suggested that at least some of its effects may be secondary to its calcium-mobilizing action.
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