Date of Award

1986

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Pharmaceutical Sciences

First Advisor

Robert Rodgers

Abstract

ENHANCED VASCULAR COLLAGEN SYNTHESIS HAS BEEN IMPLICATED AS AN ETIOLOGIC FACTOR IN THE DEVEOLPMENT OF SYSTEMIC HYPERTENSION IN VARIOUS ANIMAL MODELS . WITHIN THE AORTA AND RESISTANCE VESSELS OF SPONTANEOUSLY HYPERTENSIVE RATS ( SHR), ACCUMULATION OF FIBROUS PROTEINS (COLLAGEN, EI.ASTIN) AND SMOOOTH MUSCLE CELL HYPERPLASIA AND HYPERTROPHY ARE ASSOCIATED WITH AN INCREASE IN SYSTOLIC ARTERIAL PRESSURE (SAP). FURTHERMORE, AGENTS WHICH LOWER SAP ALSO REDUCE VASCULAR COLLAGEN SYNTHESIS. INITIAL OBSERVATIONS FROM OUR LABORATORY INDICATED THAT STREPTOZOTOCIN - INDUCED DIABETES (8 WEEKS DURATION) LOWERS SAP OF SHR WITHOUT AFFECTING SAP OF NORMOTENSIVE CONTROL WISTAR - KYOTO RATS (WKY). IN ORDER TO CHARACTERIZE THE COMPONENTS OF THIS DEPRESSOR EFFECT, I PROPOSED TO EXAMINE THE ROLE OF VASCULAR COLLAGEN BIOSYNTHESIS IN DIABETIC SPONTANEOUSLY HYPERTENSIVE RATS. THE RESULTS SHOWED THAT DIABETES LOWERED THE ACTIVITES OF MARKER ENZYMES FOR COLLAGEN BIOSYNTHESIS, PROLYL HYDROXYLASE AND LYSYL OXIDASE, IN THE SHR STRAIN AORTAE AND MESENTERIC ARTERIES .WITHOUT AFFECTING WKY ENZYME ACTIVITIES . THESE RESULTS PROVIDED PRELIMINARY EVIDENCE THAT THE DEPRESSOR EFFECT OF DIABETES MAY BE RELATED IN PART TO A REDUCTION IN VASCULAR COLLAGEN SYNTHESIS. TO FURTHER CHARACTERIZE THE APPARENT INHIBITION OF COLLAGEN SYNTHESIS, MORE DIRECT IN VITRO MEASUREMENTS WERE NECESSARY. HYDROXYPROLINE ANALYSIS IS THE STANDARD METHOD FOR QUANTITATION OF COLLAGEN SYNTHESIS AND CONTENT, DUE TO ITS ABUNDANCE IN COLLAGEN ( 107. ) AND ITS RELATIVELY INFREQUENT OCCURENCE IN OTHER PROTEINS. THE METHOD WE SELECTED FOR THE ANALYSIS OF HYDROXYPROLINE SPECIFIC ACTIVITY WAS ii REVERSE PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY, UTILIZING PRECOLUMN DERIVATIZATION WITH FLUORESCENT MARKER, 7-CHLOR0-4-NITROBENZOFURAZAN (NBD-CL). NBD-CL PREFERENTIALLY LABELS SECONDARY AMINES UNDER APPROPRIATE EXPERIMENTAL CONDITIONS. QUANTITATION OF HYDROXYPROLINE AND PROLINE STANDARDS WERE LINEAR OVER A RANGE OF AMOUNTS (4 TO 45nmol). INCORPORATION OF 14c-PROLINE INTO 14c-HYDROXYPROLINE , REPRESENTING THE RELATIVE RATE OF DE NOVO COLLAGEN SYNTHESIS , WAS LINEAR UP TO 4 AND 10 HOURS IN NONVASCULAR TUMOR HOMOGENATES AND IN AORTIC TISSUE, RESPECTIVELY. THE METHOD WAS THEN APPLIED TO QUANTIFY COLLAGEN SYNTHESIS AND CONCENTRATION FROM DIABETIC SHR AND WKY AORTAE. DIABETES MARKEDLY REDUCED SHR MESENTERIC ARTERIAL PROLYL HYDROXYLASE ACTIVITY AND AORTIC 14C-PROLINE INCORPORATION INTO 14C-HYDROXYPROLINE , IN VITRO. DIABETES ALSO INCREASED AORTIC COLLAGEN CONCENTRATION IN THE SHR STRAIN. THESE RESULTS FURTHER SUPPORT THE HYPOTHESIS THAT CHRONIC DIABETES HAS STRAIN DEPENDANT EFFECTS ON VASCULAR COLLAGEN METABOLISM. WHETHER THE OBSERVED METABOLIC CHANGES ARE EFFECTS OR CAUSES OF THE REDUCTION IN ARTERIAL PRESSURE REMAINS TO BE DETERMINED.

Share

COinS