Aortic Endothelial and Smooth MuscleHistamine Metabolism inExperimental Diabetes

Alicia Orlidge and Theodore M. Hollis

We studied histamine metabolism, i.e., histldine decarboxylase (HD)-mediated syn-thesis and hlstamlnase-mediated catabollsm, in relation to intracellular histaminecontent in both aortic endothelial and subjacent smooth muscle cells of control anddiabetic rats. Diabetes was Induced by a single jugular vein Injection of streptozotocln(55 mg/kg in acidified saline, pH 4.5), and animals were held for either 2 or 4 weeksfollowing overt manifestation of diabetes. An additional 4-week diabetic group re-ceived Insulin (lletln NPH, 10 U per 24 hour) during the last week. With respect tocontrol values, the histamine content of aortic endothelial cells increased 138%, HDactivity Increased 250%, and histaminase activity decreased 50% over the 4-weekperiod. In subjacent smooth muscle cells, the histamine content Increased In excessof 150%, HD activity Increased more than 300%, and histaminase activity decreased inexcess of 30%. Insulin treatment for the last week resulted In complete reversal of allthese changes. These results support the concept that a large vessel response similarto the microclrculatory prolonged phase of inflammation occurs In experimental dia-betes, a change similar to that occurring in experimental atherosclerosis. They alsoIndicate that both synthetic and catabollc changes occur In histamine metabolismunder these conditions, changes that alter arterlai wall histamine pools, and suggestthat Insulin administration under conditions of experlmentai diabetes may modulateaortic histamine metabolism and the resultant Intraaortlc histamine pools.(Arteriosclerosis 2: 142-150, March/April 1982)

Epidemiological data have established that dia-betes mellitus constitutes an important risk fac-

tor for atherosclerosis.1"4 Other studies have clearlyestablished that in individuals of any age athero-sclerosis is more prevalent among diabetics thannondiabetics, that these individuals show moreadvanced atherosclerosis than their nondiabeticcounterparts, and that complications of athero-sclerosis are largely responsible for the signifi-cantly higher mortality rate of diabetics.25"8 Inattempts to elucidate atherogenic mechanismsassociated with diabetes, most work has centered

From the Department of Biology, Mueller Laboratory, ThePennsylvania State University, University Park, Pennsylvania.

Supported by U.S. Public Health Service Grant HL620460.Address for reprints: Dr. Theodore M. Hollis, 208 Mueller Labo-

ratory, The Pennsylvania State University, University Park, Penn-sylvania 16802.

Received July 16, 1981; revision accepted February 1, 1982.

around characterization of alterations in lipid ca-tabolism and associated changes in circulatinglipoprotein patterns.

Only a few studies have been directed towardexamining specific metabolic changes occurringin large arteries, which are themselves directlyassociated with altered permeability, vessel sub-strate accumulation, and atherogenesis. One sig-nificant study is by Wolinsky et al.,9 who reportedthat the activities of a number of aortic smoothmuscle hydrolases are decreased in streptozoto-cin diabetes in the rat; they noted that the largestdecrease occurred in acid cholesteryl esterase,the principal enzyme involved in catabolism andclearance of low density lipoproteins (LDL). Theseresults suggest that, at least under experimentaldiabetic conditions, the capacity of arterial cells toclear LDL is impaired at the same time that theplasma LDL concentration is elevated. If this is

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AORTIC HISTAMINE METABOLISM IN DIABETES Orlidge and Hollis 143

indeed the case, then factors such as histaminewhich are intimately associated with altered wallpermeability or intravascular accumulation of ma-cromolecules, such as low density lipoprotein(LDL), could be extremely important in determin-ing the atherogenic potential of diabetes.

The present study is concerned with the charac-terization of aortic endothelial and smooth musclehistamine metabolism and intracellular histaminecontent under normal conditions as well as inexperimental diabetes. The rationale for undertak-ing these studies comes from previous work wehave performed which indicates that, under a vari-ety of atherogenic conditions, aortic histaminesynthesis is increased, that this increase is inmany cases intimately associated with an in-crease in arterial transmural uptake of circulatingalbumin,10"14 and that partial inhibition of aortic his-tamine synthesis under conditions of dietary-in-duced hyperlipidemia reduces both increases inaortic albumin uptake as well as the severity ofatherosclerosis.15

Methods

Experimental Protocol

Male Wistar rats having initial body weights of180 to 220 g (8 to 10 weeks old) were used inthese studies. Diabetes was induced by a singlejugular vein injection of streptozotocin (SigmaChemical Company, St. Louis, Missouri) at a doseof 55 mg/kg in 0.5 ml of 0.9% saline containing0.02 M sodium citrate (pH 4.5). Control animalsreceived acidified, citrated saline only. Animalswere housed in metabolic cages for daily monitor-ing of food and water intake and urine output. Uri-nary glucose was monitored using glucose oxi-dase reagent strips (Clinistix, Ames Company,Elkhart, Indiana). Serum glucose concentrationswere measured at 3 days after streptozotocin in-jection and at weekly intervals thereafter using theglucose oxidase method as adapted for a YellowSprings glucose autoanalyzer on blood obtainedfrom tail clipping. Animals were held for 2 or 4weeks following overt manifestation of diabetes,e.g., hyperglycemia, glycosuria; in the 4-weekgroup, additional rats were given 10 units of insulin(lletin NPH, Eli Lilly and Company, Indianapolis,Indiana) subcutaneously in the late afternoon dai-ly during the fourth week. Animals were killed bydecapitation, the thoracic aorta excised, and en-dothelial cells removed as described by Gospo-darowicz et al.16 Verification of isolated cell types(endothelial and smooth muscle) involved bothtransmission electron microscopy of fresh isolatesand phase contrast microscopy of primary cell cul-tures established from these cell isolates (seebelow).

Each cell type (endothelial or smooth muscle)from each aorta was placed in a separate test tubecontaining 2 ml of ice-cold phosphate-buffered sa-line (0.01 M, pH 7.0) containing 0 .1% Triton X-100and homogenized using a ground glass pestle.Then 1 ml of this homogenate was subjected toSephadex G-25 filtration (Pharmacia Fine Chemi-cals, Piscataway, New Jersey) for removal of en-dogenous substrates (such as histamine, histi-dine, and S-adenosylmethionine) which mightinfluence specific enzymatic reactions of the as-say. The collected protein fraction was stored inliquid N2 for subsequent determination of histidinedecarboxylase (EC 4.1.1.22) and histaminase(EC 1.4.3.6) activities. Histamine content was de-termined from boiled tissue homogenates to pre-vent in vitro catabolism. All measurements wereperformed at least in triplicate.

Biochemical Procedures

Tissue histamine content and histidine decar-boxylase (HD) activity were determined by modifi-cation of the double isotopic microassay of Taylorand Snyder.17 This assay uses imidazole-N-meth-yltransferase (HMT) prepared from guinea pigbrain as described by Shore.18 With [14C]-S-aden-osylmethionine ([14C]-SAMe, 58.4 nCi/mM, NewEngland Nuclear, Boston, Massachusetts) used asthe methyl donor, guinea pig brain HMT methylatessample histamine, thereby forming 1-methyl-(l3-aminoethyl)-imidazole [14C]-methylhistamine). Traceamounts of [3H] histamine (6.1 (jCi/mM, 0.64 ^Ci/ml, New England Nuclear, Boston, Massachu-setts) were added to correct for varying degrees ofmethylation among samples. With use of a stan-dard curve of known amounts of histamine, con-structed at the time of each individual assay, theratio of [14C]-dpm/[3H]-dpm gives the amount ofhistamine present per sample. Specific details ofthese assays have been described previously.19

For determination of cell histaminase activity,triplicate assays were performed with the methodof Kupfer and Roscoe.20 This assay was per-formed on supernatant not subjected to Sephadexfiltration, with the incubation medium containing0.2 ml of cell homogenate, 0.4 ml of 0.25 M phos-phate buffered saline (PBS, pH 7.5), 0.1 ml of [14C]histamine dihydrochloride (labeled in the two posi-tion on the imidazole ring; 0.25 jaCi, 0.91 pCi/nM,Amersham Corporation, Arlington Heights, Illi-nois), and 1.9 ml of distilled water. Blanks wereprepared using boiled homogenate with a 90-min-ute incubation period. Results were standardizedon the basis of protein.21

Cell Type Verification

Verification of cell types used for biochemicalevaluations involved both ultrastructural examina-tion of cell isolates and phase contrast examina-

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tion of primary cultures established from theseisolates.

For ultrastructural examination, cells were re-moved as described above and collected in coldPBS (4°C, pH 7.35). Cold glutaraldehyde wasadded to make a final concentration of 2.5%. Afterincubation for 1 hour (4°C), suspensions werecentrifuged (10,000 g, 5 min, 25°C), and the cellpellet was resuspended in 0.3 ml of agar (45°C),which was then allowed to harden on a glass co-verslip. Square blocks (1 mm2) were then cut andpostfixed with 1 % osmium tetraoxide in PBS (pH7.4) overnight, washed in PBS, and stained for 2hours in 0.1 M sodium acetate-buffered uranylacetate. Cells were dehydrated at 20-minute inter-vals in a graded series of ethanol and infiltratedwith Spurr's epoxy resin.22 Following a 12-hourincubation period at 80°C, thin sections (silver togray) were cut on a Sorval-Porter-Blum MT-2 ul-tramicrotome with glass knives, captured on a300-mesh grid, and stained for 20 minutes with3% uranylacetate in 15% methanol, followed by a2-minute staining with 0.2% lead citrate. Photomi-crographs were taken with a Hitachi 11 E trans-mission electron microscope.

Primary cultures were established using stan-dard tissue culture techniques. For endothelialcells, isolates were removed using sterile swabsand collected in Minimum Essential Medium withEarles salts (MEM, Flow Laboratories, Inc.,McLean, Virginia) containing 10% fetal bovine se-rum (FBS, Sterile Systems, Logan, Utah). Theywere plated onto sterile collagen-coated covers-lips (1 mg/ml, Ethicon Corp., Sommerville, NewJersey), and held in culture for 5 to 10 days (37°C,5% CO2) or until apparently confluent. Refeedingoccurred every 48 hours. Smooth muscle stripswere cut into 1 mm2 pieces, and explant cultureswere established in MEM-10% FBS as before.Both were then viewed using phase contrast mi-croscopy and photographed using a Nikon invert-ed phase microscope.

Statistical Analyses

To compare the differences in any metabolicparameter between a given cell type of a singleaorta, Student's nest was employed. For compari-sons of differences among treatment groups, vari-ance analysis followed by application of Duncan'smultiple range test was used, with a p value of lessthan 0.05 being considered significant. Additional-ly, linear and multiple regression analyses werealso performed to estimate dependency of hista-mine content on enzyme activity or activities.23

Results

Characteristics of Animal Groups

Final body weights, serum glucose concentra-tion, and other parameters used to monitor thediabetic state for each animal group are given intable 1. It is apparent that animals receiving strep-tozotocin exhibited a reduced rate of weight gain,hyperglycemia, increased water intake, and urineoutput. All differences between the diabeticgroups and control animals were significant (p <0.001). Insulin treatment during the 4th weekeliminated all apparent diabetic symptoms exceptthe reduced weight gain.

Ultrastructural Findings

Photomicrographs of cell isolates and resultingprimary cultures from a control animal are shownin figures 1 and 2. Figure 1 shows electron photo-micrographs of cells obtained by aortic luminalsurface scrapings (A) and by removal of subjacentmedial layers (B). Figure 2 shows phase contrastphotomicrographs of primary cultures of these cellisolates. Ultrastructural features of the luminalcells were characteristically endothelial, with nu-merous endocytic vesicles, a prominent ovoid nu-

Table 1. Characteristics of Animal Groups

Group

ControlDiabetic

2 wks)Diabetic

(4 wks)Diabetic

(3+ wks)

No.

20

10

10

10

Final bodyweight (g)

362 ± 3

238 ± 10t

243 ± 9f

240 ± 3t

Serumglucose(mg/dl)

128 ± 14

394 ± 28

414 ± 76t

85 ± 19

Urinevolume

(ml/48 hrs)

48 ± 3

342 ± 9f

325 ± 8 |

67 ± 5

Urineglucose

negative

dark*

dark

negative

H2O intake(ml/48 hrs)

99 ± 9

520 ± 16

509 ± 11

71 ± 6

All values represent means ± mean standard errors. Diabetes was induced by a single jugular veininjection of streptozotocin (55 mg/kg) in acidified citrated saline (pH 4.5).

*dark = Ames Clinistix color denoting urinary glucose concentration > 150 mg/dl.tSignificant difference from control (p < 0.001), determined by variance analysis followed by Duncan's

multiple range test.

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AORTIC HISTAMINE METABOLISM IN DIABETES Orlidge and Hollis 145

BFigure 1. Transmission electron photomicrographs of endothelial and smooth muscle cells isolated from rataorta. A. Transmission electron photomicrograph of a fresh isolate of endothelial cells; the internal marker equals 0.5urn. B. Transmission electron photomicrograph of an isolate of smooth muscle cells from the same aorta as in A;internal marker equals 1 urn.

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146 ARTERIOSCLEROSIS VOL 2, No 2, MARCH/APRIL 1982

Figure 2. Phase contrast photomicrographs of primary cultures derived from endothelial and smooth muscle cellsisolated from rat aorta. A. Phase contrast photomicrograph of a fresh isolate of endothelial cells; internal markerrepresents 0.5 nm. B. Phase contrast photomicrograph of an isolate of smooth muscle cells from the same aorta;internal marker equals 20 urn.

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AORTIC HISTAMINE METABOLISM IN DIABETES Orlidge and Hollis 147

Table 2.Diabetes

Aortic Hlstamlne Metabolism In Endothelial and Subjacent Smooth Muscle Cells After 2 Weeks of

MeanSE

Histamine

Control

EC

2.662.721.862.692.932.632.552.422.971.292.47

± 0.16 :

SMC

5.044.261.592.852.425.793.263.575.01—

3.75t± 0.30

(nmole/mg)*

Diabetic

EC

0.745.655.042.385.364.733.961.831.821.453.76*

± 0.44 :

SMC

8.599.198.187.997.989.718.536.097.577.668.22}:

t 0.32

HD Activity (nmole/hr/mg)*

Control

EC

0.4760.5500.3980.4720.6100.5680.5450.4760.6270.3760.509

SMC

0.2800.1970.1000.2290.1890.3450.1000.2010.234

—0.208t

± 0.030+ 0.030 :

Diabetic

EC

1.892.581.320.992.991.381.791.031.070.961.66*

± 0.22

SMC

1.131.271.220.890.861.381.170.640.760.791.01*

± 0.08

Histaminase

Control

EC

2847.92093.32183.62774.31699.02063.52127.92894.31602.92959.02324.0

± 159.7

SMC

814.0792.0

1918.41073.71102.1796.3

1790.5710.6821.5

—1091.0t

± 151.0

(dpm/mg)*

Diabetic

EC

1175.0911.7972.1

1281.0677.9

1194.51234.51327.41347.61459.81158.2*± 74.9

SMC

570.5346.3539.9709.9683.3287.1576.9736.8701.8677.0594.3*

± 50.2

EC = endothelial cells; SMC = smooth muscle cells; HD = histidine decarboxylase. Diabetes induced by administrationof streptozotocin (55 mg/kg, i.v.).

'Activity standardized on the basis of mg cell protein.tDifference from control EC significant (p < 0.005); determined by variance analysis followed by Duncan's multiple range

test.*Difference from control EC significant (p < 0.05).

cleus, paucity of mitochondria, and typical interen-dothelial junctions.24"26 A glycogen granule wasbeneath these cells, representing a noncellularcontaminant associated with the isolation of thesecells. The phase contrast photomicrograph of aprimary culture derived from the isolate (figure 2A) is likewise clearly endothelial. Cells shown infigures 1 B and 2 B exhibited features characteris-tic of smooth muscle cells.27

Biochemical Studies

Differences between Endothelial and SmoothMuscle Cells

The histamine content, HD, and histaminaseactivities of aortic endothelial and subjacentsmooth muscle cells from both control and 2-weekdiabetic rats are given in table 2; data for the 4-week groups are summarized in figure 3. Withrespect to the 2-week experiments, data indicatedthat under control conditions the histamine con-tent of smooth muscle cells was 52% higher (p <0.005) than that of the overlying endothelial cells.Interesting, however, was the finding that endo-thelial HD and histaminase activities were both

Figure 3. Aortic histamine metabolism in endothelialand subjacent smooth muscle cells after 4 weeks of diabe-tes with and without insulin treatment. Diabetes was in-duced by i.v. administration of streptozotocin (55 mg/kg) inacidified citrated saline (pH 4.5). Diabetic period was 4weeks. Insulin (10 U, lletin NPH) was given daily for thelast 7 days to the diabetic-insulin group only. Each groupcontained 10 rats (n = 10). EC = endothelial cells; SMC= smooth muscle cells; HD = histidine decarboxylaseactivity.

more than twice that of smooth muscle cells, i.e.,control endothelial cells had a lower histaminecontent but higher rates of synthesis and catabo-lism than did their subjacent smooth muscle cells.These differences were likewise highly significant(p < 0.005).

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Changes Occurring in Experimental Diabetes

These data also show that, under conditions ofexperimental diabetes, striking and highly signifi-cant (p < 0.005) alterations occurred in both aorticendothelial and smooth muscle histamine metab-olism over this 2-week period. Specifically, in en-dothelial cells the intracellular histamine contentwas elevated 52%, HD activity was increased by226%, with histaminase activity reduced 50%. Insmooth muscle cells from these same aortas, thehistamine content showed a 119% increase, HDactivity a 385% increase, and histaminase activitya 46% decrease.

Over the 4-week period of streptozotocin-in-duced diabetes, changes in histamine content andHD activity became significantly more pro-nounced. In endothelial cells, histamine contentwas increased over control values by 150%, withHD activity increased by 260%. In smooth muscle,histamine showed a 166% increase over controlvalues, with HD activity increasing 300%. For bothcell types, these differences between 2- and 4-week treatment groups were highly significant (p< 0.005). However, with insulin treatment for thelast 7-day period, it was apparent that a completereversal in all measured parameters occurred,since under this condition no significant differenceexisted in either endothelial or smooth muscle his-tamine content and histaminase activity with re-spect to corresponding control values. In the caseof HD activity, in both cell types this activity wassignificantly lower (p < 0.05) than that of the con-trol cells.

Statistical Analyses

A series of multiple regression analyses wereundertaken to determine which metabolic pa-rameter best predicts the observed intracellularhistamine content. In the control endothelial cells,interactions of HD-mediated synthesis and hista-minase-mediated catabolism yielded an r2 of 0.81(p < 0.001) and was described by the equationy = 0.203 + 4.93XT + 0.001 x2, where x-^ and x2represent HD and histaminase activities, respec-tively. The strongest single predictor of the hista-mine content in this cell type was the HD activity(r2 = 0.79, p < 0.001; y = 0.025 + 4.81 x). Boththe 2- and 4-week diabetic endothelial cells exhib-ited similar relationships, with 94% of the hista-mine content accounted for by combined hista-mine synthesis and catabolism. The equation wasy = 14.4 + 1.13X1 - 0.012x2 (r2 = 0.94, p <0.001), where x-i and x2 are again respective ac-tivities of HD and histaminase. Similarly, in thisdiabetic state the strongest single predictor of his-tamine content was again HD-mediated synthesis(y = -3.13 + 5.31x, r2 = 0.91, p < 0.005).

With respect to smooth muscle, under controlconditions the multiple regression equation was

y = 2.21 + 10.7X, - 0.007x2(r2 = 0.61, p < 0.025)

for these same parameters. In this case, histamin-ase activity was the strongest single predictor(y = 9.99 - 0.0096x, r2 = 0.71, p < 0.005).Interestingly, in smooth muscle cells from the 2-and 4-week diabetic groups, combined HD andhistaminase activities statistically accounted formore than 90% of the total intracellular histamine.With insulin treatment, 92% of the smooth musclehistamine was statistically accounted for by hista-minase activity alone (y = 7.57 - 0.008x, r2 =0.92, p < 0.001).

Discussion

Kahlson and Rosengren28 and Levine and Noll29

have described three metabolic pools of histaminepresent in mammalian tissues. Of these, two haveslow turnover rates and consist of inactive, boundhistamine. One of these slow turnover or inactivepools is associated with mast cells and is readilydepleted by Compound 48/80; the other is not fullycharacterized and its depletion has not yet beenachieved. Both, however, are associated with lowHD activities. The third pool, nascent or induciblehistamine, consists of unbound, HD-dependentactive histamine. Its magnitude is determined bythe balance between histamine synthesis and ca-tabolism, and is readily depleted by the inhibitionof HD. This pool has been implicated in the regula-tion of microcirculatory flow,3031 and aortic trans-mural permeability and macromolecule accumula-tion under conditions of vascular injury (see ref. 14for summary of references). In the rabbit aorta,Foldes et al.32 have shown that 37% of arterialhistamine is present in this pool; in dogs, Owensand Hollis19 have found that the magnitude of thenascent pool varies depending on aortic location,i.e., regions exposed to locally disturbed flow andthat take up Evans blue dye have significantlyhigher nascent histamine than do adjacent, whiteaortic regions.

In our present study, under normal conditionsendothelial cells have an HD activity that is almosttwice that of the underlying smooth muscle cellsand a histaminase activity that is essentially dou-ble that of these same smooth muscle cells; how-ever, the smooth muscle histamine content is 58%higher than these endothelial cells. By regressionanalyses, results indicate that more than 80% ofthe endothelial histamine content can be account-ed for on the basis of combined interactions of HDand histaminase activities, while in smooth mus-cle the most significant predictor is the rate of his-tamine catabolism. From these findings, we con-clude that a major component of endothelialhistamine appears to be contained within the na-scent histamine pool, while in smooth muscle un-der normal conditions the nascent histamine pool

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AORTIC HISTAMINE METABOLISM IN DIABETES Orlidge and Hollis 149

is relatively small; in the latter case, the majority ofintracellular histamine must be derived from someother source. Undoubtedly, for both endothelialand smooth muscle cells one such source is plas-ma, since Foldes et al.32 have shown incubatedaortic strips accumulate histamine against a hista-mine concentration gradient. For smooth muscle,we speculate that an additional and significantsource may be endothelial-derived, for while en-zymatic activities of endothelial cells indicate thatthey have a relatively rapid histamine turnover, it isalso possible that there may be significant hista-mine diffusion from both endothelium to blood andfrom endothelium to the underlying smooth mus-cle. If this is the case, these results would indicatethat under normal conditions vascular smoothmuscle could act as an intracellular histaminesink.

In the diabetic state, results of this study indi-cate that marked alterations occur in histaminecontent and histamine metabolism of both endo-thelial and underlying smooth muscle cells. Forendothelial cells, HD activity increases during the4-week period by over 260%, while histaminaseactivity is reduced by essentially 50%. The intra-cellular histamine content is increased by up to150% over control values. For smooth musclecells there is, over this period, a striking 300%increase in HD activity and up to 40% reduction inhistaminase activity. Histamine in these cells in-creases in excess of 165% over control values.Significantly, insulin treatment for a 1-week dura-tion produces complete reversal in all of theseparameters. From the statistical data it is thus ap-parent that under these diabetic conditions thereoccurs a large increase in the HD-dependent his-tamine pool of both cell types, with the largestincrease occurring in smooth muscle. Indeed, inthese cells from diabetic animals, combined hista-mine synthesis and catabolism can account for90% of the intracellular histamine present. Thatthese marked alterations in histamine metabolismare completely reversed in the insulin-treatedgroup is strong evidence that insulin treatment insome way modulates histamine metabolism andthe resultant nascent histamine pool in the aorta,and that in this respect the aorta may be consid-ered an insulin-sensitive organ, at least in experi-mental diabetes.

As previously noted, increases in aortic and aor-tic endothelial histamine synthesis occur underconditions of experimental hypertension followingexposure of the aorta to elevated shear stressesand in dietary-induced hypercholesterolemia (seeref. 14 for a summary of references). All of theseconstitute known atherogenic risk factors.34'73334

In the case of hypercholesteremia, the increase inhistamine synthesis is transient, restricted to theendothelium,3538 and is intimately associated withtransient increases in aortic transmural uptake ofcirculating albumin.14 In this case, partial inhibition

of aortic HD resulted in a 5 1 % decrease in aorticalbumin uptake and a 63% decrease in athero-sclerotic lesion severity.15 Thus, under at least ex-perimental dietary hypercholesteremia, the endo-thelial nascent histamine pool influences aorticuptake or accumulation of circulating macromole-cules. From the present study, it is apparent thathistamine synthesis is likewise increased in strep-tozotocin-induced diabetes, but with the followingimportant differences: 1) changes in histaminemetabolism occur in both endothelial and smoothmuscle cells; 2) they are of considerably greatermagnitude than those observed in dietary-inducedhypercholesteremia; 3) these changes are exac-erbated over time rather than transient.

Obviously, additional studies must be complet-ed before definitive statements can be made re-garding the true significance of the data of thepresent study. However, with previous work show-ing correlations between local vessel wall hista-mine synthesis and wall albumin accumulationunder a variety of atherogenic conditions,1114 in-cluding experimental diabetes mellitus,13 as wellas with other data clearly demonstrating reducedcapacity of the diabetic aortic smooth muscle tocatabolize internalized LDL,9 we believe that theincreased nascent histamine pool is at least onemechanism having potential for increasing therate of presentation of this substrate to underlyingwall components. If so, then altered arterial wallhistamine metabolism resulting in an increasednascent histamine pool may represent an impor-tant link with respect to the atherogenicity of dia-betes mellitus.

AcknowledgmentsThe authors are indebted to Donald M. Hessler for his technical

assistance in the preparation and evaluation of tissue sectionsusing transmission electron microscopy; to Dr. Christine Gregg forher initial critical review and constructive comments; and to Mar-garet Wolfe and Mary Alice Shea for typing this manuscript.

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Index Terms: aortic histamine metabolismhistamine metabolism • atherosclerosis •

• endothelium • smooth musclediabetes • insulin

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A Orlidge and T M HollisAortic endothelial and smooth muscle histamine metabolism in experimental diabetes.

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