THE DEPRESSOR EFFECT OF STREPTOZOTOCIN-INDUCED DIABETES IN SPONTANEOUSLY HYPERTENSIVE RATS: THE ROLE OF VASCULAR COLLAGEN SYNTHESIS

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

Recently, reverse phase high performance liquid chromatography (RP-HPLC) was applied to imino acid analysis with and without precolumn derivatization [4,5,6,7). Prior treatment of the sample with 4-chloro-7-nitrobenzofurazan (NBD-Cl) allows preferential labelling and sensitive fluorometric detection of secondary amines. This approach has been used to assay hydroxyproline in urine, plasma and purified collagen standards [4,8,9). However, it has not been employed to quantify collagen levels or synthesis rates in tissues.
The present report describes the application of RP-HPLC of NBD derivatives to determine tissue collagen levels and synthesis rates in two tissue types: rat aorta and human xenograft tumors. The values for collagen content obtained with this method correlate closely with those 3 using the Juva and Prockop procedure [10]. The method also allows the determination of specific activities of hydroxyproline and praline in each tissue sample. 4

Sample Preparation
Radiolabelled vascular tissue and tumors: Cis-4-hydr oxy -L-proline (C -4-Hyp; 5 mM, 0.020 ml) was added to each sample as an internal standard due to its convenient elution pattern between t-4-Hyp and proline. Derivatizing agent, NBD-Cl (0.5 ml of 16 mM in methanol) was added to the above mix and incubated at 60°c for 3 minutes in the dark [8,9). Collagen s ynthesis was quantified as the amount of radioactive hydroxyproline collected under the corresponding 6 peak .
Derivatization was expressed as a percentage of the total radioactivity recovered under the hydroxyproline and praline peaks. The mean derivation of radiolabelled praline and hy drox yproline in tissue samples was 90 ± 57.. All samples were corrected to 1007. for calculations of collagen content and synthesis.

Instrumentation and Chromatographic Conditions
The We sought to provide maximal derivatization of Hyp without sacrificing the selectivity of NBD-Cl for secondary amines. Increasing the time of incubation at 60°C beyond 3 minutes did not significantly increase the extent of derivatization of 14 C-proline and 3 tt-Hyp (Table I).
However, the selectivity of NBD-Cl for t-4 -Hyp diminished as time of incubation increased beyond 5 minutes. These data support previous studies, suggesting that shorter reaction times favor the selective derivatization of secondary amines [4,8,9]. Therefore, all subsequent incubations were carried out for 3 minutes at 60°C. Higher (picomolar) sensitivities could be obtained; however, this was not necessary for tissue analysis.
The separation of amino acid peaks from aortic hydrolysates was comparable to that of the corresponding standards ( Figure 2).
Therefore, prior treatment of the tissue samples with Dowex columns [13] or with o-opthalaldehyde [9,14] was not necessary.      The results demonstrated that STZ induced diabetes markedly reduced aortic collagen synthesis of SHR, but had no effect on collagen synthesis on aortae from normotensive Wistar -Kyoto rats (WKY). These data lend further support to the hypothesis that the depressor effect of diabetes in SHR may be related to reduced rates of arterial collagen synthesis. Experimental diabetes, of several weeks duration, lowers SAP of SHR without significantly influencing blood pressure of normotensive controls (8)(9)(10). A component of the depressor effect in SHR may be secondary to reductions in cardic output or pressure development ( 11).

Quantification of Collagen Content and Collagen Synthesis From
However, diabetes also disrupts vascular smooth muscle metabolism (12,13). We recently reported that streptozotocin (STZ) induced diabetes reduced aortic and mesenteric arterial prolyl hydroxylase and lysyl oxidase activity in SHR, and hypothesized that the depressor effect in this strain is associated with a reduction in collagen biosynthesis. However, more direct evidence was required to support or refute the h ypothesis.

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The purpose of the present study was to characterize the effects of diabetes

Determination of Protein, Serum Glucose and Serum Thyroxine
Proteins were quantified by the method of Lowry et. al (16), using bovine serum albumin as a protein standard.

Statistics
Mean values between groups within strains were compared us i ng the unpaired Student's t test.
Mean values between strains and other multiple comparisons were made using a non-parametric Wilcoxon sum test.

RESULTS
The effects of STZ-induced diabetes of 8 weeks duration (henceforward referred to as "diabetes") on final body weight, serum glucose and thyroxine and SAP are shown in Table 1. Diabetes reduced body weights of SHR and WKY by 48 and 367. , respectively, compared to controls of each strain.
In both strains, diabetic animals were hyperglycemic and hypothyroid. Diabetes significantly reduced SAP of SHR rats, but did not affect pressures of WKY rats.
Mesenteric arterial prolyl hydroxylase (PH) activity of SHR was higher than that of WKY controls ( The weight of rats (14 weeks old) prior to STZ injection were 259 ± 9 and 204 ± 6 for SHR and WKY, respectively.

2
Significantly different from the control of the same strain (p < 0.05).

3
Significantly different from the WKY control group. In addition, hyperglycemia can cause vasodilation and reduced vascular resistance. (37) .

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The results of this study support the hypothesis that diabetes selectively reduces vascular collagen synthesis of SllR but no t of WKY

rats.
The reduced arterial collagen synthesis ma y , in part, be responsible for the depressor effect of diabetes in SllR. Wee ks of Diabetes  Significantly different from the control of the same strain (p < 0 . 05).