Low CO2 Stimulates Inositol Phosphate Turnover and Increased Inositol 1,4,5-Trisphosphate Levels in Piglet Cerebral Microvascular Smooth Muscle Cells

Maria Luiza C. Albuquerque, Lisa Lowery-Smith, Pauline Hsu, Elena Parfenova, Charles Leffler

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Abstract

In contrast to hypercapnic dilation, hypocapnia-induced cerebral vasoconstriction does not involve prostanoids in newborn pigs. The hypothesis that increased pH or decreased CO2 tension increases inositol phosphate turnover in piglet cerebral microvascular smooth muscle (SM) cells was addressed to begin to assess the possibility that this second-messenger system is involved in hypocapnia-induced cerebral vasoconstriction. Cerebral microvascular SM cells in primary culture prelabeled with [3H]-myoinositol were stimulated for 30 sec with artificial cerebrospinal fluid of increased or normal pH, (7.80 vs 7.40), constant PCO2 36 mm Hg. Following extraction from cells, radiolabeled inositol phosphates were separated by HPLC. These metabolic alkalosis studies were repeated using an inositol 1,4,5-trisphosphate (lns[1,4,5]P3 protein-binding assay (PBA). Respiratory alkalosis using aCSF with pH 7.60, PCO2 20 mm Hg versus control pH 7.40, PCO2 36 mm Hg was similarly tested with PBS measurement of Ins(1,4,5)P3. aCSFs of control pH 7.40, and PCO2s of 70, 36, or 25 mm Hg were studied both by [3H]-myoinositol (HPLC) and PBA to further determine the importance of CO2 tension, in the presence of fixed pH, on Ins(1,4,5)P3 production. When PCO2 was constant, inositol phosphate turnover (as measured by [3H]-lns[1,4,5]P3 accumulation) increased when pH was increased from 7.40 to 7.80 at 30 sec of stimulation. Mean [3H]-Ins(1,4,5)P3 accumulation at pHs of 7.40 and 7.80, constant PCO2 of 36 mm Hg, were 2.9 ± 0.7 and 4.1 ± 0.8 cpm/μg protein, respectively. Ins(1,4,5)P3 levels for pH of 7.40 or 7.80 and constant PCO2 of 36 mm Hg, were 25.4 ± 1.8 and 38 ± 8 pmol/well, respectively, by PBA. Respiratory alkalosis also increased Ins(1,4,5)P3 levels. For pH of 7.40, PCO2 36 mm Hg and pH 7.60, PCO2 20 mm Hg, Ins(1,4,5)P3 levels were 37.6 ± 16 and 64.1 ± 25 pmol/well, respectively. Decreasing CO2 tension (from 70 mm Hg to 25 mm Hg) in the presence of fixed pH 7.40 failed to increase Ins(1,4,5)P3 levels. The present data demonstrate that decreased CO2 tension stimulates an increase in Ins(1,4,5)P3 production in piglet cerebral microvascular smooth muscle cells. Increasing pH via lower PCO2 increases the level of Ins(1,4,5)P3 even more than increasing pH with fixed base, but extracellular pH appears to be important since decreased PCO2 without changing extracellular pH had no effect. We conclude that the inositol phosphate second messenger system in cerebral microvascular smooth muscle responds appropriately to acute alkalosis to be involved in hypocapnia-induced cerebral vasoconstriction.

Original languageEnglish (US)
Pages (from-to)14-19
Number of pages6
JournalProceedings of the Society for Experimental Biology and Medicine
Volume209
Issue number1
DOIs
StatePublished - Jan 1 1995

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Inositol 1,4,5-Trisphosphate
Inositol Phosphates
Smooth Muscle Myocytes
Muscle
Cells
Assays
Second Messenger Systems
Inositol
Cerebrospinal fluid
Hypocapnia
Prostaglandins
Vasoconstriction
Protein Binding
Respiratory Alkalosis
Alkalosis
Proteins
High Pressure Liquid Chromatography

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

@article{c114358c47c34e229db6a3c115decfc4,
title = "Low CO2 Stimulates Inositol Phosphate Turnover and Increased Inositol 1,4,5-Trisphosphate Levels in Piglet Cerebral Microvascular Smooth Muscle Cells",
abstract = "In contrast to hypercapnic dilation, hypocapnia-induced cerebral vasoconstriction does not involve prostanoids in newborn pigs. The hypothesis that increased pH or decreased CO2 tension increases inositol phosphate turnover in piglet cerebral microvascular smooth muscle (SM) cells was addressed to begin to assess the possibility that this second-messenger system is involved in hypocapnia-induced cerebral vasoconstriction. Cerebral microvascular SM cells in primary culture prelabeled with [3H]-myoinositol were stimulated for 30 sec with artificial cerebrospinal fluid of increased or normal pH, (7.80 vs 7.40), constant PCO2 36 mm Hg. Following extraction from cells, radiolabeled inositol phosphates were separated by HPLC. These metabolic alkalosis studies were repeated using an inositol 1,4,5-trisphosphate (lns[1,4,5]P3 protein-binding assay (PBA). Respiratory alkalosis using aCSF with pH 7.60, PCO2 20 mm Hg versus control pH 7.40, PCO2 36 mm Hg was similarly tested with PBS measurement of Ins(1,4,5)P3. aCSFs of control pH 7.40, and PCO2s of 70, 36, or 25 mm Hg were studied both by [3H]-myoinositol (HPLC) and PBA to further determine the importance of CO2 tension, in the presence of fixed pH, on Ins(1,4,5)P3 production. When PCO2 was constant, inositol phosphate turnover (as measured by [3H]-lns[1,4,5]P3 accumulation) increased when pH was increased from 7.40 to 7.80 at 30 sec of stimulation. Mean [3H]-Ins(1,4,5)P3 accumulation at pHs of 7.40 and 7.80, constant PCO2 of 36 mm Hg, were 2.9 ± 0.7 and 4.1 ± 0.8 cpm/μg protein, respectively. Ins(1,4,5)P3 levels for pH of 7.40 or 7.80 and constant PCO2 of 36 mm Hg, were 25.4 ± 1.8 and 38 ± 8 pmol/well, respectively, by PBA. Respiratory alkalosis also increased Ins(1,4,5)P3 levels. For pH of 7.40, PCO2 36 mm Hg and pH 7.60, PCO2 20 mm Hg, Ins(1,4,5)P3 levels were 37.6 ± 16 and 64.1 ± 25 pmol/well, respectively. Decreasing CO2 tension (from 70 mm Hg to 25 mm Hg) in the presence of fixed pH 7.40 failed to increase Ins(1,4,5)P3 levels. The present data demonstrate that decreased CO2 tension stimulates an increase in Ins(1,4,5)P3 production in piglet cerebral microvascular smooth muscle cells. Increasing pH via lower PCO2 increases the level of Ins(1,4,5)P3 even more than increasing pH with fixed base, but extracellular pH appears to be important since decreased PCO2 without changing extracellular pH had no effect. We conclude that the inositol phosphate second messenger system in cerebral microvascular smooth muscle responds appropriately to acute alkalosis to be involved in hypocapnia-induced cerebral vasoconstriction.",
author = "Albuquerque, {Maria Luiza C.} and Lisa Lowery-Smith and Pauline Hsu and Elena Parfenova and Charles Leffler",
year = "1995",
month = "1",
day = "1",
doi = "10.3181/00379727-209-43871",
language = "English (US)",
volume = "209",
pages = "14--19",
journal = "Experimental Biology and Medicine",
issn = "1535-3702",
publisher = "SAGE Publications Ltd",
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TY - JOUR

T1 - Low CO2 Stimulates Inositol Phosphate Turnover and Increased Inositol 1,4,5-Trisphosphate Levels in Piglet Cerebral Microvascular Smooth Muscle Cells

AU - Albuquerque, Maria Luiza C.

AU - Lowery-Smith, Lisa

AU - Hsu, Pauline

AU - Parfenova, Elena

AU - Leffler, Charles

PY - 1995/1/1

Y1 - 1995/1/1

N2 - In contrast to hypercapnic dilation, hypocapnia-induced cerebral vasoconstriction does not involve prostanoids in newborn pigs. The hypothesis that increased pH or decreased CO2 tension increases inositol phosphate turnover in piglet cerebral microvascular smooth muscle (SM) cells was addressed to begin to assess the possibility that this second-messenger system is involved in hypocapnia-induced cerebral vasoconstriction. Cerebral microvascular SM cells in primary culture prelabeled with [3H]-myoinositol were stimulated for 30 sec with artificial cerebrospinal fluid of increased or normal pH, (7.80 vs 7.40), constant PCO2 36 mm Hg. Following extraction from cells, radiolabeled inositol phosphates were separated by HPLC. These metabolic alkalosis studies were repeated using an inositol 1,4,5-trisphosphate (lns[1,4,5]P3 protein-binding assay (PBA). Respiratory alkalosis using aCSF with pH 7.60, PCO2 20 mm Hg versus control pH 7.40, PCO2 36 mm Hg was similarly tested with PBS measurement of Ins(1,4,5)P3. aCSFs of control pH 7.40, and PCO2s of 70, 36, or 25 mm Hg were studied both by [3H]-myoinositol (HPLC) and PBA to further determine the importance of CO2 tension, in the presence of fixed pH, on Ins(1,4,5)P3 production. When PCO2 was constant, inositol phosphate turnover (as measured by [3H]-lns[1,4,5]P3 accumulation) increased when pH was increased from 7.40 to 7.80 at 30 sec of stimulation. Mean [3H]-Ins(1,4,5)P3 accumulation at pHs of 7.40 and 7.80, constant PCO2 of 36 mm Hg, were 2.9 ± 0.7 and 4.1 ± 0.8 cpm/μg protein, respectively. Ins(1,4,5)P3 levels for pH of 7.40 or 7.80 and constant PCO2 of 36 mm Hg, were 25.4 ± 1.8 and 38 ± 8 pmol/well, respectively, by PBA. Respiratory alkalosis also increased Ins(1,4,5)P3 levels. For pH of 7.40, PCO2 36 mm Hg and pH 7.60, PCO2 20 mm Hg, Ins(1,4,5)P3 levels were 37.6 ± 16 and 64.1 ± 25 pmol/well, respectively. Decreasing CO2 tension (from 70 mm Hg to 25 mm Hg) in the presence of fixed pH 7.40 failed to increase Ins(1,4,5)P3 levels. The present data demonstrate that decreased CO2 tension stimulates an increase in Ins(1,4,5)P3 production in piglet cerebral microvascular smooth muscle cells. Increasing pH via lower PCO2 increases the level of Ins(1,4,5)P3 even more than increasing pH with fixed base, but extracellular pH appears to be important since decreased PCO2 without changing extracellular pH had no effect. We conclude that the inositol phosphate second messenger system in cerebral microvascular smooth muscle responds appropriately to acute alkalosis to be involved in hypocapnia-induced cerebral vasoconstriction.

AB - In contrast to hypercapnic dilation, hypocapnia-induced cerebral vasoconstriction does not involve prostanoids in newborn pigs. The hypothesis that increased pH or decreased CO2 tension increases inositol phosphate turnover in piglet cerebral microvascular smooth muscle (SM) cells was addressed to begin to assess the possibility that this second-messenger system is involved in hypocapnia-induced cerebral vasoconstriction. Cerebral microvascular SM cells in primary culture prelabeled with [3H]-myoinositol were stimulated for 30 sec with artificial cerebrospinal fluid of increased or normal pH, (7.80 vs 7.40), constant PCO2 36 mm Hg. Following extraction from cells, radiolabeled inositol phosphates were separated by HPLC. These metabolic alkalosis studies were repeated using an inositol 1,4,5-trisphosphate (lns[1,4,5]P3 protein-binding assay (PBA). Respiratory alkalosis using aCSF with pH 7.60, PCO2 20 mm Hg versus control pH 7.40, PCO2 36 mm Hg was similarly tested with PBS measurement of Ins(1,4,5)P3. aCSFs of control pH 7.40, and PCO2s of 70, 36, or 25 mm Hg were studied both by [3H]-myoinositol (HPLC) and PBA to further determine the importance of CO2 tension, in the presence of fixed pH, on Ins(1,4,5)P3 production. When PCO2 was constant, inositol phosphate turnover (as measured by [3H]-lns[1,4,5]P3 accumulation) increased when pH was increased from 7.40 to 7.80 at 30 sec of stimulation. Mean [3H]-Ins(1,4,5)P3 accumulation at pHs of 7.40 and 7.80, constant PCO2 of 36 mm Hg, were 2.9 ± 0.7 and 4.1 ± 0.8 cpm/μg protein, respectively. Ins(1,4,5)P3 levels for pH of 7.40 or 7.80 and constant PCO2 of 36 mm Hg, were 25.4 ± 1.8 and 38 ± 8 pmol/well, respectively, by PBA. Respiratory alkalosis also increased Ins(1,4,5)P3 levels. For pH of 7.40, PCO2 36 mm Hg and pH 7.60, PCO2 20 mm Hg, Ins(1,4,5)P3 levels were 37.6 ± 16 and 64.1 ± 25 pmol/well, respectively. Decreasing CO2 tension (from 70 mm Hg to 25 mm Hg) in the presence of fixed pH 7.40 failed to increase Ins(1,4,5)P3 levels. The present data demonstrate that decreased CO2 tension stimulates an increase in Ins(1,4,5)P3 production in piglet cerebral microvascular smooth muscle cells. Increasing pH via lower PCO2 increases the level of Ins(1,4,5)P3 even more than increasing pH with fixed base, but extracellular pH appears to be important since decreased PCO2 without changing extracellular pH had no effect. We conclude that the inositol phosphate second messenger system in cerebral microvascular smooth muscle responds appropriately to acute alkalosis to be involved in hypocapnia-induced cerebral vasoconstriction.

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