Date of Award

Spring 1986

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry & Biochemistry

Program/Concentration

Chemistry

Committee Director

Michael J. Dimino

Committee Member

Robert L. Ake

Committee Member

Michael C. Beatrice

Committee Member

Roy L. Williams

Call Number for Print

Special Collections LD4331.C45S22

Abstract

Luteinizing hormone (LH) increases intracellular concentrations of adenosine 3', 5 '-cyclic monophosphate and the phosphoinositides, phosphatidylinositol (PI), PI 4'-phosphate (PIP) and PI 4' .5 1 - bispbosphate (PIP2). It is believed that cAMP and the phosphoinositides act concertedly to regulate mitochondrial conversion of cholesterol to pregnenolone. This study examined the effects of LH and N6 ,O2 -dibutyryl cAMP (dbcAMP) on phospholipids metabolism by luteal mitochondria and the influence of dbcAMP and the phosphoinosi tides on mitochondrial steroid production. Mitochondria were isolated from unincubated and incubated luteal tissue by differential centrifugation. Phospholipids were extracted from the mitochondria with an acidified CHCl3:CH3OH mixture, separated by two dimensional thin layer chromatography (2D-TLC) and quantitated by analysis for phosphate. Steroids were extracted from mitochondria with petroleum ether and quantitated by radioimmunoassay (RIA).

Mitochondria isolated from uninoubated luteal tissue contained 305 nmoles phospbolipids/mg protein. When luteal tissue was incubated in Medium 199D (0.3g/ml} without or with 30 μ M dbcAMP, total mitochondrial phospholipids decreased. Incubation of luteal tissue with 1 μg/ml LH maintained the mitochondrial phospholipid concentration at levels equivalent to those found in unincubated tissue. LH also caused the synthesis of PI and phosphatidic acid (PA).

Incubation of isolated from untreated luteal tissue increased total phospholipid extracted. Mitochondria incubated in the presence of 30 μM dboAMP showed a decrease in PI and an increase in PA concentrations with respect to mitochondria incubated without dbcAMP. Incubated mitochondria were shown to perform oxidative phosphorylation and synthesize steroids. Addition of the phosphoinositides did not affect the ability of the mitochondria to synthesize adenosine 5 'triphosphate (ATP) but increased mitochondrial ateroidogenesia. Mitochondria incubated with 2 μM PIP or PIP2 synthesized significantly more steroid than untreated or PI treated mitochondria after 15 min of incubation. Mitochondria incubated with 2 μM dbcAMP and 2 μM of a phosphoinositide synthesized significantly more steroids than mitochondria incubated with either dbcAMP or a phosphoinositide alone after 5 min of incubation.

These results indicate that in luteal mitochondria, LH causes an increase in PI concentration by a cyclic nucleotide independent event. cAMP causes a catabolism of the accumulated PI. We hypothesized that the catabolism of PI occurred via the phosphoinositide pathway. That is to say, PI is phosphorylated stepwise to PIP and PIP2 and the latter compound is degraded to 1,2-diacyl-sn-glycerol {diacylglycerol) and inositol 1,4,5-trisphosphate. It is likely that the catabolism of PIP2 is the step regulated by cAMP.

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DOI

10.25777/yd6n-5s97

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