EDRF released from microvascular endothelial cells dilates arterioles in vivo.

EDRF released from microvascular dilates arterioles in vivo A. KOLLER, Department

N. SEYEDI,

M. E. GERRITSEN,

AND

cells

G. KALEY

of Physiology, New York Medical College, Valhalla, New York 10595

KOLLER, A.,N. SEYEDI,M.E. GERRITSEN,ANDG.KALEY. EDRF released from microvascular endothelial cells dilates arterioles in vivo. Am. J. Physiol. 261 (Heart Circ. Physiol. 30): H128-H133, 1991.-Microvascular endothelial cells (MECs) from rat epididymal fat pad were isolated and cultured in vitro on Cytodex 3 microcarrier beads. In Krebs-suffused cremaster muscle of pentobarbital-anesthetized rats arteriolar diameters (mean control diam 20.9 t 0.9 pm) were measured using image shearing video microscopy. Two lines of suffusate (1.5 ml/min each) were established; one contained a column of microcarrier beads only (no cells in line; NC) the other contained a l-ml column of MECs grown on beads (through cells; TC). The muscle preparation and the MECs were first treated with indomethacin (Indo; 28 PM). Indo treatment blocked arteriolar dilation to A23187 (1 PM) and arachidonic acid (AA; 0.25 FM) administered into the NC line. A 4.0 t 0.6 pm increase in arteriolar diameter was observed, however, when A23187 (but not AA) was infused through the TC line containing Indotreated MECs on beads. The A23187-elicited dilation was abolished by the introduction of NG-monomethyl-L-arginine (LNMMA; 200 PM) into the TC line. Administration of atropine (2 PM) onto the cremaster muscle via the NC line inhibited the dilations in response to acetylcholine (ACh; 2.7 PM) given through the NC line. Infusion of ACh through the TC line onto the atropine-treated cremaster muscle, however, elicited a 5.8 t 1.3 pm increase in arteriolar diameter, a response that was blocked by prior administration of L-NMMA into the TC line. Arteriolar dilation induced by adenosine (0.5 PM) or sodium nitroprusside (0.5 PM) applied via the NC or TC line was unaffected by L-NMMA. Results of our experiments suggest that cultured microvascular endothelial cells are able to release in basal conditions and upon stimulation by agonists, a nonprostaglandin vasodilator principle that has the attributes of endothelium-derived relaxing factor. bioassay; cell culture; endothelium-derived vasodilator factors; prostaglandins; acetylcholine; A23187; skeletal muscle microcirculation; indomethacin; N”-monomethyl+arginine

MICROVASCULAR ENDOTHELIAL CELLS (MECs) have been previously demonstrated to produce a variety of prostaglandins (7, 9). However, vascular endothelium is capable of synthesizing other factors that relax vascular smooth muscle (l-3, 5, 6, 22). Bioassay experiments using large vessels with endothelium (6, 27, 30) gave evidence for the synthesis of a nonprostanoid vasorelaxant, namely endothelium-derived relaxing factor (EDRF). Cultured endothelial cells from large vessels have also been demonstrated to produce EDRF (l-3, 19, 2O), now believed to be nitric oxide or a nitroso compound derived from L-arginine, whose synthesis is blocked by H128

endothelial

NG-monomethyl+arginine ( L-NMMA) (13, 23-26, 28). Our previous in vivo studies (14-17) suggested that the endothelium of skeletal muscle arterioles may be involved in the dilation of arterioles to acetylcholine via production of a vasoactive substance that has the attributes of EDRF, as characterized in large arteries. A recent study also showed that a labile factor is released from bovine aortic endothelial cells upon exposure to A23187 or bradykinin and that it elicits dilation of arterioles of the hamster cheek pouch (26). It is known that there are inherent differences among endothelial cells derived from various vascular beds and those from vessels of different size (6,7). However, whether a nonprostanoid dilator factor is actually produced by cultured microvascular endothelial cells in response to the calcium ionophore A23187 and acetylcholine (substances known to release EDRF from large vessels) and whether it can affect microvessels in vivo has not yet been demonstrated. The aim of the present study was, therefore, to investigate whether cultured MECs from rat epididymal fat pad produce EDRF in response to these two agonists, whether the EDRF released is capable of dilating arterioles (-20 pm) of rat cremaster muscle in vivo, and whether it is synthesized via L-arginine. METHODS Cremaster muscle preparation. Male Wistar rats 5-6 wk of age were anesthetized with a subcutaneous injection of pentobarbital sodium (35 mg/kg). A constant level of anesthesia was maintained throughout the experiment by subcutaneous injection of supplemental doses (20% of original dose) of the anesthetic agent every 30-45 min. The trachea was cannulated to facilitate respiration. Arterial blood pressure was monitored with a Statham P23 D6 transducer connected to a cannula inserted in the left common carotid artery and recorded on a Sensormedics Dynograph recorder (model R 5llA). The left cremaster muscle was prepared with caution to maintain an intact blood and nerve supply (16). Throughout the surgical procedure the muscle was kept moist and warm. The temperatures of the animal and the supporting Plexiglas platform for the muscle were thermostatically controlled at 37 and 33.5”C, respectively. During the experiment a Krebs solution (33.5”C) composed of 154 mM NaCl, 5.6 mM KCl, 2.2 mM CaC12, 1.2 mM MgSO,, and 20 mM NaHC03 and equilibrated with ambient air superfused the cremaster muscle via two lines (see below). Use of the PO:! of ambient air

0363-6135/91 $1.50 Copyright 0 1991 the American Physiological Society

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MICROVASCULAR

EDRF

permitted the concurrent study of isolated endothelial cells and cremaster muscle and afforded sufficient arteriolar tone to examine dilator responses. The pH of the solution was maintained at 7.35 with sodium bicarbonate and hydrochloric acid. These conditions provided for a stable preparation throughout the course of the experiments (14, 16, 17). The image of the cremaster microvessels was visualized with television microscopy (Olympus, Lake Success, NY) using a ~20 objective and recorded on a video tape. Transillumination with a tungsten lamp was used for observation and measurement of diameter. The resting internal diameter of arterioles and changes in diameter in response to vasoactive agents were measured with an image-shearing monitor (model 908, Instruments for Physiology and Medicine, San Diego, CA). Vessels selected for study were third-order arterioles, 20.9 t 0.9 pm in diameter. Preparation of microvascular endothelial cells. Microvascular endothelial cells (MECs) were isolated from rat epididymal fat pads (8, 29, 31) and cultured essentially as described by Madri and Williams (21). Microvascular tufts, a mixture of arterioles, venules, and capillaries were plated on 35mm culture plates precoated with 2% gelatin (Eastman Kodak, Rochester, NY). MECs were grown in RPM1 containing 20% fetal bovine serum, and 2 mM glutamine supplemented with endothelial cell growth factor (75 pg/ml) and heparin (20 pg/ml), prepared as described previously (8, 9). Cultures were incubated in a 5% C02-95% air humidified atmosphere at 37°C. MECs were passaged at confluence with 0.05% trypsin in edatate sodium (Versene) and split at a 1:4 ratio. Criteria for the endothelial nature of these cells included virtually 100% positive staining using fluorescein isothiocyanate-conjugated antisera to factor VIIIrelated antigen (Atlantic Antibodies, Scarborough, ME); avid incorporation of l,l’-dioctadecyl-1,3,3’,3’-tetramethylindocarbocyanine perchlorate-acetylated low density lipoprotein (Biomedical Technologies, Staughton, MA); and positive staining with antisera against angiotensin-converting enzyme (provided by Dr. Randall Skidgel, Chicago, IL). For studies of EDRF release, logphase cultures were seeded to Cytodex 3 microcarrier beads (Pharmacia AB, Uppsala, Sweden) and used within l-2 days of attaining near complete coverage of the available bead surface. Microcarrier cultures were maintained in stir bottles using a Techne microcarrier apparatus (Princeton, NJ). Cells used in this study were l-5 passages from primary isolation. At the start of the experiments, after several washes with Krebs solution, a l-ml column of MEC on beads (-2 x lo7 cells) was placed in a plastic tube (through cells; TC line) (Evergreen Scientific). The filter disk in that tube was made thinner by cutting it in half to facilitate flow. As a control, microcarrier beads without MECs were placed in another tube (no cells; NC line). Outflow from the tubes was directed over the cremasteric arteriole under study (Fig. 1). This experimental arrangement allowed the evaluation of arteriolar effects mediated directly by agonists or the suffusion solution, versus those indirectly via MEC-derived mediators. The viabilitv of the MECs in this environment was demon-

DILATES

H129

ARTERIOLES

/

solution

OBJECTIVE Drugs

ASTER MICROSCOPE 0 l

BEADS

MUSCLE

STAGE ONLY

MICROVASCULAR CELLS ON BEADS

ENDOTHELIAL

FIG. 1. Schematic arrangement of experimental system. Two suffusion lines were used. NC, Krebs superfusion solution with no microvascular endothelial cells in line (open circles); TC, Krebs superfusion solution through cultured microvascular endothelial cells (closed circles) derived from rat epididymal fat pad. A layer of Krebs solution was always present on top of the muscle preparation. See METHODS for further details.

strated by the ir continuing ability to respond to various agents as well asl from the fact that after a 6-h incubation in the media the maj ority of the cells rem .ained attached to the microcarrier beads and appeared . normal when examined by phase microscopy. A 2-ml fluid column provided a gravity-driven Krebs perfusion of both lines (Fig. 1). To these columns vasoactive substances were administered in a volume of 200 ~1 in either of the lines to reach the MEC on microcarrier beads or only microcarrier beads, without interrupting the flow of the suffusion fluid. The rate of flow in each suffusion line was 1.5 ml/min and was kept constant throughout the experiments. The “transit time” between the MECs and cremaster muscle was 5-8 s as assessed by administration of Evans blue. The resistance to flow in the NC line was adjusted to be comparable to that in the TC line by placing cotton on the disk. The tip of the plastic tubes was in contact with the surface of the fluid layer, providing for continuous flow. In preliminary experiments, we observed that this contact between the tip of pipette containing MECs and the tissue was critical (most likely because of the labile nature of the factor being transferred) to achieve arteriolar dilation in response to A23187 and acetylcholine. Initially, after surgery, Krebs solution was suffused (at 3 ml/min) via the NC line onto the muscle for 30-40 min to obtain a steady-state level of arteriolar tone. At this point, half the suffusion was diverted to the MEC-containing line (TC), an arrangement that was maintain .ed throughout the experiment, and -10 min was allowed to reach a new steady-state level of arteriolar tone. Indomethacin (28 PM) was then introduced in both lines for 30 min, followed by one of two experimental protocols. (The concentrations of agonists noted below are the presumed concentrations in the suffusion fluid on the tissue.) In six rats, the peak responses of arterioles to arachidonic acid (0.25 ,uM), A23187 (1 PM) and adenosine (0.5 PM) via the NC or TC line were determined. L-NMMA (200 PM) was then introduced in the TC line for 25 min, and the protocol was repeated. In five separate rats, arteriolar responses to arachidonic acid and acetvlcholine (0.27 uM) via the NC line


H130

MICROVASCULAR

EDRF

were determined. After atropine administration through the NC line (2 PM for 15 min), arteriolar responses to acetylcholine (2.7 PM) via the NC or TC line were tested. The effect of L-NMMA on the arteriolar responses to acetylcholine and sodium nitroprusside (0.5 PM) via the TC line was then determined. A period of 5 min between administration of each agent was used to allow the vessel to return to its control diameter. Atropine, acetylcholine chloride, adenosine, A23187, and sodium nitroprusside were obtained from Sigma Chemical (St. Louis, MO). Arachidonic acid was purchased from Nuchek (Elysian, MN) and L-NMMA (sulfonate salt) from Calbiochem (La Jolla, CA). Indomethacin was provided by Merck Sharp & Dohme (Rahway, NJ). Appropriate dilutions of substances were made with Krebs buffer before their administration. A stock solution of A23187 was prepared in dimethyl sulfoxide (DMSO), and appropriate dilutions were made in Krebs buffer. The final concentration of diluted DMSO was 0.1%. Preliminary studies indicated that administration of drug carrier solutions had no significant effect on vascular diameter. All cell culture supplies were purchased from GIBCO (Grand Island, NY). Data are reported as means t SE with n indicating the number of an imals. In each experimental animal only one vessel was studied. Statistical analysis was performed using analysis of variance and t test. A P value of ~0.05 was considered significant. RESULTS

Mean arterial blood pressure of rats was stable throughout the experiments and was within 95-115 mmHg. During the initial part of the experiments when the suffusion of muscle was only via the NC line, basal arteriolar diameter was 20.9 t 0.9 pm (Table 1). Diverting half the Krebs solution through the TC line resulted in a significant arteriolar dilation to 27.6 t 1.5 pm (P < 0.05). This increase in diameter was significantly reduced (to 21.8 t 1.2 pm) when indomethacin was added to the suffusion solution. The efficacy of cyclooxygenase inhibition was confirmed by the absence of vasodilator responses to arachidonic acid. In the later phase of the experiments when, additionally, L-NMMA was added to the suffusion, a further significant reduction (to 15.2 t 1.7 pm) in basal arteriolar diameter was observed.

DILATES

ARTERIOLES

The calcium ionophore A23187 is a potent stimulator of both prostaglandin (9) and EDRF release (6) from endothelial cells. In a previous study, we demonstrated that in rat cremaster muscle the arteriolar dilation in response to topical application of A23187 was entirely mediated by prostaglandins (14). In the present study indomethacin was added to the suffusion solution to block prostaglandin synthesis in MECs and in the arterioles of cremaster muscle. Under these circumstances A23187 applied through the NC line did not elicit arteriolar dilation. However, when A23187 was administered through the TC line, a vasodilator response was observed in spite of the presence of indomethacin (Fig. 2). This indomethacin-resistant component of the A23187-elicited vasodilation was abolished when L-NMMA was added to the Krebs solution suffusing the TC column (Fig. 3). Dilator responses of the arteriole to adenosine were not affected by the various experimental manipulations. In the course of the next series of experiments, our purpose was to investigate further the nature of the EDRF-like factor produced by cultured MECs. Thus the effects of acetylcholine, administered through the TC and NC lines, were compared (Fig. 4). Acetylcholine (0.27 F Through

Beads

Only

Through

L-NM?(IAY

Cells On Beads

tr

-g

30

k +

*O

“5

10

a

-E

0

.

-

-F--.-

m

e

l

l

.

! Pmin

t

t

A23187

ADO

f

t

t

A23187

A23187

ADO

FIG. 2. Representative record of the effect of A23187 (1 PM) and adenosine (ADO; 0.5 PM) on diameter of a cremasteric arteriole (in the presence of 28 PM indomethacin) administered either through line containing only beads, or through the line containing cultured microvascular endothelial cells derived from rat epididymal fat pad on beads. Then N”-monomethyl-L-arginine ( L-NMMA; 200 PM) was introduced in the through cell superfusion line, and the effects of A23187 and ADO were reexamined.

lc3

T

pm

cus-1

J---THR~UGH

CELLS+

1. Changes in arteriolar diameter in response to vasoactive factors from microvascular endothelial cells TABLE

Perfusion

NC TC TC TC

line line line + Indo line + Indo

Arteriolar

+ L-NMMA

Diameter,

pm

20.9t0.9 27.6&G* 21.8t1.2* 15.2a.7*t

Summary data (means + SE, n = 11) of effect of diverting Krebs suffusion solution through microvascular endothelial cells derived from rat epididymal fat pad on the basal diameter of cremasteric arterioles in the presence of indomethacin (Indo, 28 PM) and Indo plus NCmonomethyl-L-arginine (L-NMMA, 200 PM) in the suffusion solution. NC line, no cells in line; TC line, through cells in line. * P < 0.05, change in arteriolar diameter vs. the previous value; t P < 0.05 vs. NC line.

T 7-

p

A23

ADO

AA

A23

I--L-NMMA-I A23 ADO

FIG. 3. Summary of A23187 (A23; 1 PM)-induced release of EDRF from cultured microvascular endothelial cells from rat epididymal fat pad. Adenosine (ADO; 0.5 PM); NG-monomethyl+arginine, L-NMMA (200 PM). Lack of arteriolar response to arachidonic acid (AA; 0.25 PM) signifies effective cyclooxygenase blockade by indomethacin (28 PM). Values are means & SE; n = 6. * P < 0.05 vs. baseline diameter.


MICROVASCULAR + Through

Beads

c

Only

ATROPINE

I

Through

*

’

EDRF

L-NMMA----i

Cells

On

Beads 1

c

1

2min t ACH

t

t

ACH

t

t

ACH

ACH

SNP

FIG. 4. Representative record of changes in diameter of a cremasteric arteriole first to acetylcholine (ACh) administered via the line containing only beads, before (0.27 PM) and after (2.7 PM) the muscle was treated with atropine (2 PM). Then ACh (2.7 PM) was administered via the line containing cultured microvascular endothelial cells, in the presence of atropine (on muscle only) and in the additional presence of N”-monomethyl-L-arginine (L-NMMA; 200 PM) given via the through cell line. SNP, sodium nitroprusside (0.5 PM). Throughout the 1 experiments the superfusion solution contained 28 PM indomethacir to inhibit prostaglandin synthesis in the cells and in cremaster muscle

I---NO

CELLS ----I

I-THROUGH

I-L-NMMA-(

I-ATROP-) AA

ACH

ACH

SNP

CELLS-

ACH

ACH

SNP

FIG. 5. Summary of acetylcholine (ACh)-induced release of EDRF from cultured microvascular endothelial cells derived from rat epididymal fat pad. The effect of ACh was first tested via the line containing no cells before (0.27 PM) and after (2.7 PM) the administration of atropine (Atrop; 2 PM) on the muscle. Then the effects of ACh administered via the line containing the cells were retested first in the absence and then in the presence of N”-monomethyl-L-arginine (LNMMA; 200 PM). SNP, sodium nitroprusside (0.5 PM). Lack of response to arachidonic acid (AA; 0.25 PM) signifies effective cyclooxygenase blockade by indomethacin (28 PM). Values are means t SE; n = 5. * P < 0.05 vs. baseline diameter.

PM) administered via the NC line elicited an indomethacin-resistant vasodilation. This arteriolar response was abolished by atropine applied to the cremaster muscle (via the NC line). Atropine itself did not cause a significant change of arteriolar diameter. In contrast, when acetylcholine was administered through the TC line it elicited an arteriolar dilation despite the presence of atropine on the muscle. After the MECs were suffused with L-NMMA the acetylcholine-elicited vasodilation was completely inhibited (Fig. 5). The arteriolar response to sodium nitroprusside was not, however, affected by any of the experimental interventions. DISCUSSION

The present study demonstrates that micro vascular endothelial cells deri ved from rat epidi .dymal fat pad

DILATES

ARTERIOLES

H131

release vasodilator factors in response to A23187 and acetylcholine, which are known to elicit EDRF production from endothelial cells of large vessels. As an on-line in vivo bioassay for MEC-derived dilator factors, we have used the suffused rat cremaster muscle microcirculation, monitoring the diameter of a thirdorder arteriole. The vasodilator nature of the effluent from MECs was evidenced by its ability to elicit a significant (32%) increase in arteriolar diameter. In these conditions indomethacin (through MECs) caused a much greater reduction in diameter (24.3%) than when indomethacin was applied directly onto the muscle [ 12.6 (17) and 11.9% (15)]. These findings together suggest that one of the MEC-derived vasodilator factors, released under basal conditions, is prostanoid in nature. This notion also corresponds with the observation that MECs synthesize both prostaglandins Ez and I2 (M. E. Gerritsen and N. Seyedi; unpublished observations). We also found that the application of L-NMMA via MECs resulted in a greater reduction (30.3%) in basal arteriolar diameter than when the inhibitor was applied directly on the muscle (15.5%) (15). Because L-NMMA is a competitive inhibitor of nitric oxide synthase (13, 23, 25, 28), these findings suggest that the nonprostanoid dilator factor released from MECs is most likely EDRF. Thus it seems that in these experimental conditions, when MECs are exposed to constant flow of superfusion fluid, they release both prostaglandins and EDRF. A23187 has been shown previously to elicit prostanoidand nonprostanoid-mediated relaxation of large blood vessels and to evoke the release of EDRF from bovine aortic (24) and human umbilical endothelial cells (30). Work by several groups has also suggested that a nonprostanoid vasodilator substance can be released by MECs (5, 10, 11, 27). Rivers et al. (26) found recently that A23187 stimulates the release of a labile transferable factor from cultured bovine aortic endothelial cells that elicits arteriolar dilation in the hamster cheek pouch. Because direct application of A23187 onto the vessels of the hamster cheek pouch had only minimal effects and because the cells were treated with indomethacin, the dilator factor released, they conclude, was most likely EDRF. In our previous studies we found, however, that topical application of A23187 to rat cremaster muscle arterioles in vivo elicited a significant vasodilation that, contrary to the above-mentioned studies, was mediated entirely by prostaglandins, since its action was completely blocked by indomethacin (14). At the present time, it remains unclear why A23187 elicits vasodilation in the intact microcirculation entirely via the release of prostaglandins, whereas exposure of MECs to A23187 stimulates the release of both prostaglandins (9) and EDRF. One explanation could be that MECs from fat pad are a mixture of arteriolar, capillary, and venular endothelial cells, which can have different capacities to produce EDRF. When applied directly, acetylcholine evoked vasodilation of skeletal muscle arterioles that was blocked by atropine applied onto the muscle. However, when acetylcholine was suffused through MECs it evoked the release of a nonprostanoid vasodilator factor whose action was not blocked by the presence of atropine on the


H132

MICROVASCULAR

EDRF

cremaster muscle. Acetylcholine was shown previously to induce calcium transients in primary cultures of rabbit aortic endothelial cells (4). Increases in calcium efflux from bovine MECs in response to ATP were also observed, although the relaxant activity produced in response to ATP by rabbit aortic spiral strips (denuded of endothelium) was minuscule (27). In in vivo bioassay experiments that used a pial window preparation in the cat (18), acetylcholine elicited the release of a labile transferable factor that had properties quite similar to those of EDRF. Moreover, freshly harvested porcine aortic endothelial cells have been shown to release in response to acetylcholine a labile nonprostanoid relaxant factor (11) that causes vasodilation in the isolated perfused rabbit heart (12). The experiments described in the present paper demonstrate for the first time that EDRF is released from cultured MECs in response to acetylcholine. Methodological differences, or the different vascular source of endothelial cells, may explain the absence of muscarinic receptors on cultured endothelial cells in previous experiments (l-3, 6, 19, 20). In the present study the action of acetylcholine was blocked by treatment of MECs with L-NMMA. We have also shown recently that arteriolar dilation in rat cremaster muscle to direct topical application of acetylcholine, in contrast to that of A23187, is mediated via the L-arginine pathway, since it was nearly abolished by L-NMMA (15). Furthermore, indomethacin or L-NMMA treatment of skeletal muscle does not alter vasodilator responses to adenosine or sodium nitroprusside (15). Taken together, these observations indicate that the ability of L-NMMA to block the response of atropine-pretreated arterioles to acetylcholine in the present experiments was due to the inhibitory action of L-NMMA on EDRF synthesis in cultured MECs. It is of note that in the present study the origin of endothelial cells was the same species as the tissue on which it was tested. The hierarchical origin of the cells was also similar to the vessels used in assaying the products (i.e., microvascular). The activity of EDRF released in response to acetylcholine is in accord with our previous findings showing that arteriolar dilation to acetylcholine is dependent on the endothelium in skeletal muscle microcirculation (14-17). The successful transfer of this vasoactive factor from cultured MECs to arterioles verifies its humoral nature. At the same time these experiments are also indicative of the fact that in microvessels, as in large arteries, acetylcholine can affect vascular smooth muscle via the endothelium without any direct contact between endothelial cells and the subendothelial layer. In conclusion, these studies demonstrate that cultured MECs are able to synthesize in addition to prostaglandins a nonprostanoid vasoactive factor. The greatly augmented synthesis of this factor by the calcium ionophore A23187 in a nonspecific manner, and by acetylcholine via a receptor-mediated process, could be completely inhibited by L-NMMA. This MEC-derived factor dilates arterioles of skeletal muscle at a time when the direct effects of A23187 and acetylcholine are blocked by specific inhibitors. Based on the results of the present study, we suggest that the vasodilator factor released from

DILATES

ARTERIOLES

indomethacin-treated MECs under basal and agoniststimulated conditions is similar to or identical with the EDRF (nitric oxide or a nitroso compound) released from large vessels. By the same token, our results also suggest that EDRF could be involved in the regulation of the skeletal muscle microcirculation. The authors acknowledge the superior secretarial help of Annette Ecke and the innovative engineering help of Stefan Pischinger. This work was supported by American Heart Association New York Affiliate Grant 89-062G and by National Heart, Lung, and Blood Institute (NHLBI) Grant PO-l-HL-43023. During the period of this study, M. E. Gerritsen was supported by an NHLBI Research Career Development Award. Address for reprint requests: A. Koller, Dept. of Physiology, New York Medical College, Valhalla, NY 10595. Received

31 January

1991; accepted

in final

form

12 March

1991.

REFERENCES 1. ANGUS, J. A., AND T. M. COCKS. The half-life of endotheliumderived relaxing factor released from bovine aortic endothelial cells in culture. J. Physiol. Lond. 388: 71-81, 1987. 2. BOULANGER, C., H. HENDRICKSON, R. R. LORENZ, AND P. M. VANHOUTTE. Release of different relaxing factors by cultured porcine endothelial cells. Circ. Res. 64: 1070-1078, 1989. 3. COCKS, T. M., J. A. ANGUS, J. H. CAMPBELL, AND G. R. CAMPBELL. Release and properties of endothelium-derived relaxing factor (EDRF) from endothelial cells in culture. J. Cell. Physiol. 123: 310-320,1985. 4. DANTHULURI, N. R., M. I. CYBULSKY, AND T. A. BROCK. AChinduced calcium transients in primary cultures of rabbit aortic endothelial cells. Am. J. Physiol. 255 (Heart Circ. Physiol. 24): H1549-H1553,1988. 5. F~RSTERMANN, U., U. ALHEID, C. DUDEL, W. G. EISERT, T. H. MULLER, AND J. C. FROLICH. Production of endothelium-derived relaxing factor by microvascular endothelial cells. In: Resistance Arteries, edited by W. Halpern, B. L. Pegram, J. E. Brayden, K. Mackey, M. K. McLaughlin, and G. 0~01. Ithaca, NY: Perinatology, 1988, p. 17-24. 6. FURCHGOTT, R. F. Role of endothelium in responses of vascular smooth muscle. Circ. Res. 53: 557-573, 1983. 7. GERRITSEN, M. E. Functional heterogeneity of vascular endothelial cells. Biochem. PharmacoZ. 36: 2701-2711, 1987. 8. GERRITSEN, M. E., W. CARLEY, AND A. J. MILICI. Microvascular endothelial cells: isolation, identification and cultivation. Adu. Cell Cult. 6: 35-67, 1988. M. E., AND C. D. CHELI. Arachidonic acid and pros9. GERRITSEN, taglandin endoperoxide metabolism in isolated rabbit and coronary microvessels and isolated and cultivated microvessel endothelial cells. J. CZin. Inuest. 72: 1658-1671, 1983. 10. GLOVER, W. E., R. M. MARKS, AND G. G. PETRENAS. Evidence for the release of a vascular relaxing factor from cultured human endothelial cells. In: Vasodilatation, Vascular Smooth Muscle, Peptides, Autonomic Nerves and Endothelium, edited by P. M. Vanhoutte. New York: Raven, 1988, p. 421-425. 11. HARTMANN, A., M. SAEED, AND R. J. BING. Release of endothelium-derived relaxing factor from freshly harvested porcine endothelial cells. Circ. Res. 61: 548-554, 1987. 12. HARTMANN, A., M. SAEED, M. METZ, AND R. J. BING. Effect of EDRF release from freshly harvested endothelial cells on the coronary circulation of the isolated working rabbit heart. Microcirc. Endothelium Lymphatics 21-44, 1988. 13. IGNARRO, L. J., G. M. BUGA, K. S. WOOD, R. E. BYRNES, AND G. CHAUDHURI. Endothelium derived relaxing factor produced and released from artery and vein is nitric oxide. Proc. Natl. Acad. Sci. USA 84: 9265-9269,1987. 14. KALEY, G., J. M. RODENBURG, E. J. MESSINA, AND M. S. WOLIN. Endothelium-associated vasodilators in rat skeletal muscle microcirculation. Am. J. Physiol. 256 (Heart Circ. Physiol. 25): H720H725,1989. 15. KOLLER, A., AND G. KALEY. Prostaglandins mediate arteriolar


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16.

17.

18.

19.

20.

21.

22.

23.

EDRF

dilation to increased blood flow velocity in skeletal muscle microcirculation. Circ. Res. 67: 529-534, 1990. KOLLER, A., E. J. MESSINA, M. S. WOLIN, AND G. KALEY. Effects of endothelial impairment on arteriolar dilator responses in vivo. Am. J. Physiol. 257 (Heart Circ. Physiol. 26): H1485-H1489, 1989. KOLLER, A., E. J. MESSINA, M. S. WOLIN, AND G. KALEY. Endothelial impairment inhibits prostaglandin and EDRF-mediated arteriolar dilation in vivo. Am. J. Physiol. 257 (Heart Circ. Physiol. 26): H1966-H1970,1989. KONTOS, H. A., E. P. WEI, AND J. J. MARSHALL. In vivo bioassay of endothelium-derived relaxing factor. Am. J. Physiol. 255 (Heart Circ. Physiol. 24): H1259-H1262, 1988. LOEB, A. L., R. A. JOHNS, P. MILNER, AND M. J. PEACH. Endothelium-derived relaxing factor in cultured cells. Hypertension Dallas 9, Suppl. III: 111-186-111-192, 1987. LUCKHOFF, A., R. BUSSE, I. WINTER, AND E. BASSENGE. Characterization of vascular relaxant factor released from cultured endothelial cells. Hypertension Dallas 9: 295-303, 1987. MADRI, J. A., AND S. K. WILLIAMS. Capillary endothelial cell cultures: phenotypic modulation by matrix components. J. Cell Biol. 97: 153-165, 1983. MILNER, P. G., N. J. Izzo, JR., J. SAYE, A. L. LOEB, R. A. JOHNS, AND M. J. PEACH. Endothelium-dependent relaxation is independent of arachidonic acid release. J. Clin. Inuest. 81: 1795-1803,1988. MONCADA, S., R. M. PALMER, AND R. J. GRYGLEWSKI. Mechanism of action of some inhibitors of endothelium-derived relaxing factor. Proc. Natl. Acad. Sci. USA 83: 9164-9168, 1986.

DILATES

ARTERIOLES

H133

24. MYERS, P. R., R. GUERRA, JR., AND D. G. HARRISON. Release of NO and EDRF from cultured bovine aortic endothelial cells. Am. J. Physiol. 256 (Heart Circ. Physiol. 25): H1030-H1037, 1989. 25. PALMER, R. M. J., A. G. FERRIGE, AND S. MONCADA. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature Lord. 327: 524-526, 1987. 26 RIVERS, R. J., A. L. LOEB, N. J. Izzo, JR., M. J. PEACH, AND B. R. DULING. Microcirculatory responses to exogenous endothelial cell-derived relaxing factor. Am. J. Physiol. 258 (Heart Circ. Physiol. 27): H606-H609, 1990. 27 . RYAN, U. S., M. K. GLASSBERG, AND A. JOHNS. Signal transduction pathways in endothelial cells. In: Endothelium-Deriued Relaxing Factors, edited by G. M. Rubanyi and P. M. Vanhoutte. Basel: Karger, 1990, p. 99-116. I., D. STUEHR, D. D. GROSS, C. NATHAN, AND R. LEVI. 28* SAKUMA, Identification of arginine as a precursor of endothelium-derived relaxing factor. Proc. Natl. Acad. Sci. USA 85: 8664-8667, 1988. W. P. FAULK, AND E. C. LEROY. 2g* SHERER, G. K., T. P. FITZHARRIS, Cultivation of microvascular endothelial cells from human preputial skin. In Vitro 16: 675-684, 1980. AND I. LEUSEN. Release 30. VAN DE VOORDE, J., H. VANDERSTICHELE, of endothelium-derived relaxing factor from human umbilical vessels. Circ. Res. 60: 517-522, 1987. 31. WAGNER, R. C., AND M. A. MATTHEWS. The isolation and culture of capillary endothelium from epididymal fat. Microuasc. Res. 10: 286-297,1975. l


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