Transport in rat vessel walls. I. Hydraulic conductivities of the aorta, pulmonary artery, and inferior vena cava with intact and denuded endothelia

In this study, filtration flows through the walls of the rat aorta, pulmonary artery (PA), and inferior vena cava (IVC), vessels with very different susceptibilities to atherosclerosis, were measured as a function of transmural pressure (DeltaP), with intact and denuded endothelium on the same vessel. Aortic hydraulic conductivity (L(p)) is high at 60 mmHg, drops approximately 40% by 100 mmHg, and is pressure independent to 140 mmHg. The trends are similar in the PA and IVC, dropping 42% from 10 to 40 mmHg and flat to 100 mmHg (PA) and dropping 33% from 10 to 20 mmHg and essentially flat to 60 mmHg (IVC). Removal of the endothelium renders L(p)(DeltaP) flat: it increases L(p) of the aorta by approximately 75%, doubles L(p) of the PA, and quadruples L(p) of the IVC. Specific resistance (1/L(p)) of the aortic endothelium is approximately 47% of total resistance; i.e., the endothelium accounts for approximately 47% of the DeltaP drop at 100 mmHg. The PA value is 55% at >40 mmHg, and the IVC value is 23% at 10 mmHg. L(p) of the intact aorta, PA, and IVC are order 10(-8), 10(-7), and 5 x 10(-7) cm.s(-1).mmHg(-1), and wall thicknesses are 145.8 microm (SD 9.3), 78.9 microm (SD 3.3), and 66.1 microm (SD 4.1), respectively. These data are consistent with the different wall structures of the three vessels. The rat aortic L(p) data are quantitatively consistent with rabbit L(p)(DeltaP) (Tedgui A and Lever MJ. Am J Physiol Heart Circ Physiol 247: H784-H791, 1984; Baldwin AL and Wilson LM. Am J Physiol Heart Circ Physiol 264: H26-H32, 1993), suggesting that intimal compression under pressure loading may also play a role in L(p)(DeltaP) in these other vessels. Despite very different driving DeltaP, nominal transmural water fluxes of these three vessels are very similar and, therefore, cannot alone account for their differences in disease susceptibility. The different fates of macromolecular tracers convected by these water fluxes into the walls of these vessels may account for this difference.     

Onset of pulsatile pressure causes transiently increased filtration through artery wall

Convective fluid motion through artery walls aids in the transvascular transport of macromolecules. Although many measurements of convective filtration have been reported, they were all obtained under constant transmural pressure. However, arterial pressure in vivo is pulsatile. Therefore, experiments were designed to compare filtration under steady and pulsatile pressure conditions. Rabbit carotid arteries were cannulated and excised from male New Zealand White rabbits anesthetized with pentobarbitol sodium (30 mg/kg i.v. administered). Hydraulic conductance was measured in cannulated excised rabbit carotid arteries at steady pressure. Next, pulsatile pressure trains were applied within the same vessels, and, simultaneously, arterial distension was monitored using Optical coherence tomography (OCT). For each pulse train, the volume of fluid lost through filtration was measured (subtracting volume change due to residual distension) and compared with that predicted from steady pressure measurements. At 60- and 80-mmHg baseline pressures, the experimental filtration volumes were significantly increased compared with those predicted for steady pressure (P < 0.05). OCT demonstrated that the excess fluid volume loss was significantly greater than the volume that would be lost through residual distension (P < 0.05). After 30 s, the magnitude of the excess of fluid loss was reduced. These results suggest that sudden onset of pulsatile pressure may cause changes in arterial interstitial hydration.     

Characteristics of myogenic reactivity in isolated rat mesenteric veins

Mechanisms of mechanically induced venous tone and its interaction with the endothelium and key vasoactive neurohormones are not well established. We investigated the contribution of the endothelium, l-type voltage-operated calcium channels (L-VOCCs), and PKC and Rho kinase to myogenic reactivity in mesenteric vessels exposed to increasing transmural pressure. The interaction of myogenic reactivity with norepinephrine (NE) and endothelin-1 (ET-1) was also investigated. Pressure myography was used to study isolated, cannulated, third-order rat mesenteric small veins and arteries. NE and ET-1 concentration response curves were constructed at low, intermediate, and high transmural pressures. Myogenic reactivity was not altered by nitric oxide synthase inhibition with N(ω)-nitro-L-arginine (L-NNA; 100 μM) or endothelium removal in both vessels. L-VOCCs blockade (nifedipine, 1 μM) completely abolished arterial tone, while only partially reducing venous tone. PKC (chelerythrine, 2.5 μM) and Rho kinase (Y27632, 3 μM) inhibitors largely abolished venous and arterial myogenic reactivity. There was no significant difference in the sensitivity of NE or ET-1-induced contractions within vessels. However, veins were more sensitive to NE and ET-1 when compared with corresponding arteries at low, intermediate, and high transmural pressures, respectively. These results suggest that 1) myogenic factors are important contributors to net venous tone in mesenteric veins; 2) PKC and Rho activation are important in myogenic reactivity in both vessels, while l-VOCCs play a limited role in the veins vs. the arteries, and the endothelium does not appear to modulate myogenic reactivity in either vessel type; and 3) mesenteric veins maintain an enhanced sensitivity to NE and ET-1 compared with the arteries when studied under conditions of changing transmural distending pressure.     

Experimental determination and mathematical model of the transient incorporation of cholesterol in the arterial wall

Experimental data of the radial incorporation of labeled cholesterol [¹⁴C-4] into the artery wall is regressed against a mathematical model that predicts macromolecular transport in this biological system. Data is obtained using excised canine carotid arteries that are perfusedin vitro under pulsatile hemodynamic conditions for 2 hr. Vessels are exposed to either normotensive hemodynamics, hypertensive hemodynamics, or simulations in which the rate of flow or vessel compliance is deliberately altered. Several arteries are studied under normotensive conditions following balloon catheter deendothelialization. Transmural concentration profiles of [¹⁴C-4] activity are determined by microcryotomy of longitudinal sections of perfused vessels. Nonlinear Marquardt regression on 12 experimental cases yields parameter estimates of effective diffusivity,D and solute filtration velocity,V. Results of this experimental investigation support our hypothesis that hemodynamics and the endothelial lining influence wall flux in intact vessels. Exposure to altered (vs normotensive) hemodynamics is associated with increased incorporation of labeled cholesterol. A similar observation is made for deendothelialized vessels (e.g. a greater accumulation of label and a rise in convective flux). Based upon our companion measurements of vessel wall forces and endothelial cellular morphology accompanying hemodynamic simulations, we suggest that hemodynamically induced alterations to endothelial structures lead to the increased permeability, convection and incorporation that we observe in this work.     

Effect of cyclic axial stretch of rat arteries on endothelial cytoskeletal morphology and vascular reactivity

Pulsatile fluid shear stress and circumferential stretch are responsible for the axial alignment of vascular endothelial cells and their actin stress fibers in vivo. We studied the effect of cyclic alterations in axial stretch independent of flow on endothelial cytoskeletal organization in intact arteries and determined if functional alterations accompanied morphologic alterations. Rat renal arteries were axially stretched (20%, 0.5 Hz) around their in vivo lengths, for up to 4h. Actin stress fibers were examined by immunofluorescent staining. We found that cyclic axial stretching of intact vessels under normal transmural pressure in the absence of shear stress induces within a few hours realignment of endothelial actin stress fibers toward the circumferential direction. Concomitant with this morphologic alteration, the sensitivity (log(EC(50))) to the endothelium-dependent vasodilator (acetylcholine) was significantly decreased in the stretched vessels (after stretching -5.15+/-0.79 and before stretching -6.71+/-0.78, resp.), while there was no difference in sodium nitroprusside (SNP) sensitivity. There was no difference in sensitivity to both acetylcholine and SNP in time control vessels. Similar to cultured cells, endothelial cells in intact vessels subjected to cyclic stretching reorganize their actin filaments almost perpendicular to the stretching direction. Accompanying this morphological alteration is a loss of endothelium-dependent vasodilation but not of smooth muscle responsiveness.     

Calculation of the reflection coefficient from measurements of endogenous vascular indicators

The solvent drag reflection coefficient (sigma) for total proteins can be estimated by comparing the relative degrees of concentration of erythrocytes and plasma proteins that occur during fluid filtration in an isolated perfused organ. In this analysis, we evaluated the accuracy of equations proposed by Pilati and Maron [Am. J. Physiol. 247 (Heart Circ. Physiol. 16): H1-H7, 1984] and Wolf et al. [Am. J. Physiol. 253 (Heart Circ. Physiol. 22): H194-H204, 1987] to calculate sigma from these concentration changes. We calculated sigma with each equation using data generated from a mathematical model of fluid and solute flux in membranes with known sigma's. We found that the equation of Wolf et al. provided the closest approximation to the true sigma over the entire range of filtration fractions tested (0.1-0.6), with the differences between the two equations increasing with filtration fraction. At low filtration fractions, the difference in sigma obtained using either approach was found to be inconsequential. At larger filtration fractions, a closer approximation of the true sigma can be obtained using the equation of Wolf et al.     

Endothelin and nitric oxide mediate reduced myogenic reactivity of small renal arteries from pregnant rats

We tested the hypothesis that endothelin acting through the endothelial ET(B) receptor subtype and the nitric oxide (NO) pathway accounts for reduced myogenic reactivity of the renal resistance vasculature during pregnancy. Small renal arteries (100-200 microm) were isolated from virgin and midterm pregnant rats when gestational renal hyperfiltration and vasodilation are maximal in this species. Myogenic reactivity (the adjustment of arterial diameter in response to a change in transmural pressure) was assessed with a pressurized myograph system. A rapid increase in transmural pressure from 60 to 80 mmHg resulted in a 2.4% diameter increase in vessels from virgin compared with an 8.1% increase in arteries from midgestation rats (n = 8 each, P < 0.05). Thus myogenic reactivity is markedly reduced during pregnancy. Incubation with the NO synthase inhibitors, an ET(B) receptor subtype antagonist (RES-701-1), the nonselective ET(A/B) receptor blocker (SB-209670), or endothelial removal abrogated the reduced myogenic reactivity of vessels from gravid rats without affecting myogenic reactivity in arteries from virgin animals. Thus the endothelium mediates the reduced myogenic reactivity of small renal arteries of midgestation rats most likely through the ET(B) receptor subtype and NO pathway.     

Nitric oxide from endothelium and smooth muscle modulates responses to sympathetic nerve stimulation: implications for endotoxin shock.

The influence of nitric oxide (NO) on vascular responses to transmural stimulation (TNS) of noradrenergic nerves was studied in isolated rings of rat iliac arteries. TNS produced frequency-dependent contractions in all vessels. The NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) significantly enhanced TNS responses in intact vessels, but not in those in which the endothelium had been removed. However, in endothelium-denuded rings incubated for 8 hours, L-NMMA increased the contractions induced by nerve stimulation, an effect which was prevented by treatment with dexamethasone or cycloheximide, and enhanced by incubation with lipopolysaccharide and gamma-interferon. Addition of L-arginine reversed the effect of L-NMMA in intact rings; however, it significantly decreased below control values TNS-induced contractions in vessels without endothelium. The results indicate that a) the arterial response to noradrenergic nerve stimulation is modulated by NO originating either in endothelial cells or in smooth muscle cells after induction of NO synthase activity, and b) once NO synthase is induced, the limiting step in NO production is the availability of the substrate L-arginine. An overproduction of vascular NO in the presence of endotoxin or other inflammatory stimuli may prevent the vascular response to sympathetic stimuli and contribute to the vasodilation observed in inflammation or endotoxic shock.     

Calcitonin gene-related peptide vasodilation of human pulmonary vessels

Human calcitonin gene-related peptide (CGRP) is localized to sensory neurons in pulmonary vessels and is a potent vasodilator. We have characterized the effects of CGRP in human pulmonary vessels and localized the receptors for this peptide by autoradiography. Fresh human lung tissue was obtained from eight patients undergoing surgery and small (200-400 microns ID) pulmonary arteries and veins were dissected free of surrounding connective and pulmonary tissue. Pairs of vessels were studied and in one of each pair the endothelium was left intact and from the other of each pair the endothelium was removed by gentle abrasion. For functional studies arteries (n = 9) and veins (n = 9) were suspended in an organ bath, precontracted with 1 microM prostaglandin F2 alpha. CGRP (10 pM to 10 microM) was added in a cumulative manner. CGRP caused a dose-dependent relaxation of endothelium intact human pulmonary arteries and veins with log EC50 values of -8.01 +/- 0.35 and -8.70 +/- 0.40, respectively (not significant). Removal of the endothelium did not diminish the vasodilator potency of CGRP in either vessel. For autoradiographic studies, cryostat sections of the small human pulmonary vessels with or without endothelium were used. 125I-CGRP densely labeled CGRP receptors on vascular smooth muscle and endothelial removal did not have any effect on grain density. We concluded that CGRP is a potent vasodilator of human pulmonary arteries and veins that is not dependent on an intact endothelium. These functional studies correlate with the distribution of CGRP receptors as localized by autoradiography.     

Cilostazol improves endothelium-derived hyperpolarizing factor-type relaxation in mesenteric arteries from diabetic rats

We previously reported that in mesenteric arteries from streptozotocin (STZ)-induced diabetic rats that 1) endothelium-derived hyperpolarizing factor (EDHF)-type relaxation is impaired, possibly due to a reduced action of cAMP via increased phosphodiesterase 3 (PDE3) activity (Matsumoto T, Kobayashi T, and Kamata K. Am J Physiol Heart Circ Physiol 285: H283-H291, 2003) and that 2) PKA activity is decreased (Matsumoto T, Wakabayashi K, Kobayashi T, and Kamata K. Am J Physiol Heart Circ Physiol 287: H1064-H1071, 2004). Here we investigated whether chronic treatment with cilostazol, a PDE3 inhibitor, improves EDHF-type relaxation in mesenteric arteries isolated from STZ rats. We found that in such arteries 1) cilostazol treatment (2 wk) improved ACh-, A-23187-, and cyclopiazonic acid-induced EDHF-type relaxations; 2) the ACh-induced cAMP accumulation was transient and sustained in arteries from cilostazol-treated STZ rats; 3) the EDHF-type relaxation was significantly decreased by a PKA inhibitor in the cilostazol-treated group, but not in the cilostazol-untreated group; 4) cilostazol treatment improved both the relaxations induced by cAMP analogs and the PKA activity level; and 5) PKA catalytic subunit (Cat-alpha) protein was significantly decreased, but the regulatory subunit RII-beta was increased (and the latter effect was significantly decreased by cilostazol treatment). These results strongly suggest that cilostazol improves EDHF-type relaxations in STZ rats via an increase in cAMP and PKA signaling.     

Mechanical buckling of artery under pulsatile pressure

Tortuosity that often occurs in carotid and other arteries has been shown to be associated with high blood pressure, atherosclerosis, and other diseases. However the mechanisms of tortuosity development are not clear. Our previous studies have suggested that arteries buckling could be a possible mechanism for the initiation of tortuous shape but artery buckling under pulsatile flow condition has not been fully studied. The objectives of this study were to determine the artery critical buckling pressure under pulsatile pressure both experimentally and theoretically, and to elucidate the relationship of critical pressures under pulsatile flow, steady flow, and static pressure. We first tested the buckling pressures of porcine carotid arteries under these loading conditions, and then proposed a nonlinear elastic artery model to examine the buckling pressures under pulsatile pressure conditions. Experimental results showed that under pulsatile pressure arteries buckled when the peak pressures were approximately equal to the critical buckling pressures under static pressure. This was also confirmed by model simulations at low pulse frequencies. Our results provide an effective tool to predict artery buckling pressure under pulsatile pressure.     

Hemodynamics and the vascular endothelial cytoskeleton

Although there is considerable evidence to suggest that hemodynamics play an important role in vascular disease processes, the exact mechanisms are unknown. With this in mind, we have designed a pulsatile perfusion apparatus which reproducibly delivers pulsatile hemodynamics upon freshly excised canine carotid arteries in vitro. Quantifiable simulations included normotension with normal or lowered flow rates (120/80 mmHg, 120 and 40 ml/min), normotension with lowered or elevated transmural pressures (40-170 mmHg), and elevated pulse pressure (120 and 80 mmHg) with normal (150 ml/min) or elevated rates of flow (300 and 270 ml/min). Arterial biomechanical stresses and cellular behaviors were characterized biochemically and morphologically under all these stimulations which continued for 2-24 h. We found that increased pulse pressure alone had little effect on the total amount of radiolabeled [4-14C]cholesterol present within the medial compartment. However, normotension when coupled with altered transmural pressure yielded a three- to fourfold increase. Combinations of increased pulse pressure and flow potentiated cholesterol uptake by a factor of 10 when compared with normotension control values. Simulations that enhanced carotid arterial cholesterol uptake also influenced the endothelial cytoskeletal array of actin. Stress fibers were not present within the carotid endothelial cells of either the sham controls or the normotension and increased pulse pressure (normal flow) simulations. Endothelial cells lining carotids exposed to elevations in flow or those present within vessels perfused as per simulation b above assembled stress fibers (x = 4 and 10 per cell, respectively) within the time course of these studies. When endothelial cells were subjected to hemodynamic conditions that potentiated maximally cholesterol transport, no diffuse or stress fiber staining could be seen, but the cortical array of actin was intact. These results suggest that those biomechanical stresses that alter endothelial permeability and intimal integrity may do so via cytoskeletal actin signaling.     

Hypoxic constriction of porcine distal pulmonary arteries: endothelium and endothelin dependence

To determine the role of endothelium in hypoxic pulmonary vasoconstriction (HPV), we measured vasomotor responses to hypoxia in isolated seventh-generation porcine pulmonary arteries < 300 microm in diameter with (E+) and without endothelium. In E+ pulmonary arteries, hypoxia decreased the vascular intraluminal diameter measured at a constant transmural pressure. These constrictions were complete in 30-40 min; maximum at PO(2) of 2 mm Hg; half-maximal at PO(2) of 40 mm Hg; blocked by exposure to Ca(2+)-free conditions, nifedipine, or ryanodine; and absent in E+ bronchial arteries of similar size. Hypoxic constrictions were unaltered by indomethacin, enhanced by indomethacin plus N(G)-nitro-L-arginine methyl ester, abolished by BQ-123 or endothelial denudation, and restored in endothelium-denuded pulmonary arteries pretreated with 10(-10) M endothelin-1 (ET-1). Given previous demonstrations that hypoxia caused contractions in isolated pulmonary arterial myocytes and that ET-1 receptor antagonists inhibited HPV in intact animals, our results suggest that full in vivo expression of HPV requires basal release of ET-1 from the endothelium to facilitate mechanisms of hypoxic reactivity in pulmonary arterial smooth muscle.     

The contractile response of vascular smooth muscle to thrombin and its inhibition by thrombin inhibitors

Thrombin (0.1-30 NIH-U/ml) caused a contractile response of rabbit aortic rings. The vasocontraction was independent upon intact endothelium, however in deendothelialized vessels the contractile effect was more pronounced. The thrombin-induced vasocontraction was diminished in calcium-free medium; the same effect was attained by the calcium channel blocker verapamil at high concentrations. In human femoral and saphenous vein strips thrombin produced a contractile effect, too, which was very low and inconsistent in femoral arterial strips. To inhibit the contractile response of vascular smooth muscle to thrombin, higher concentrations of both the specific tight-binding inhibitors hirudin and beta Nas-Gly-(pAM)Phe-Pip were required than for the inhibition of fibrinogen clotting.     

Neurogenic contractions of the rat tail artery under isobaric conditions: effect of transmural pressure and function of the endothelium]

In stimulation of the rat nerve with a modulated sine pattern, an increase in the modulating frequency from 0.03 to 0.15 Hz diminished the latency between the stimulating signals and changes in the vessel resistance as well as the amplitude of the flow oscillations, but did not affect tonic contractions of the vessel. A reduction of transmural pressure from 80 to 40 mm Hg increased both the tonic and the phasic components of the vessel contraction. Following the endothelium removal no change in the response latency occurred. The data obtained suggest that, during a rhythmic neurogenic influence, the vascular endothelium may work as an "amplifier" of the vessel's phasic contractions.     

Contractile and relaxant responses of diabetic dog femoral arteries

Strips of femoral arteries of normal and alloxan-treated dogs were set up for isometric recording. The contractile response to phenylephrine and the relaxant response to acetylcholine were determined. Neither alloxan treatment nor mechanical removal of endothelium altered the EC50 value for phenylephrine. The slope of phenylephrine dose-response curves of diabetic and healthy vessels with intact endothelium was similar, whereas the slope of phenylephrine dose-response curves of endothelium-denuded diabetic arteries was significantly greater than that of the denuded healthy arteries. Removal of the endothelium completely abolished the relaxant effect of acetylcholine. The relaxant potency (IC50) of acetylcholine was not affected by alloxan treatment. The results suggest that in canine femoral arteries the relaxant activity of acetylcholine is unaffected in experimental diabetes and the damage of the arterial endothelium may play a role in the increased responsiveness of diabetic vessels to adrenergic agonists.     

Nature of pulmonary hypertension in canine oleic acid pulmonary edema

It has recently been suggested that pulmonary hypertension secondary to oleic acid lung injury mainly results from an increase in the critical closing pressure of the pulmonary vessels [Boiteau et al., Am. J. Physiol. 251 (Heart Circ. Physiol. 20): H1163-H1170, 1986]. To further test this hypothesis, we studied 1) the pulmonary arterial pressure- (Ppa) flow (Q) relationship with left atrial pressure (Pla) kept constant (n = 7) and 2) the Ppa-Pla relationship with Q kept constant (n = 9) in intact anesthetized and ventilated dogs before and after lung injury induced by oleic acid (0.09 ml/kg iv). Q was manipulated by use of a femoral arteriovenous bypass and a balloon catheter inserted in the inferior vena cava. Pla was manipulated with a balloon catheter placed by thoracotomy in the left atrium. Ppa-Q plots were rectilinear before as well as after oleic acid. Before oleic acid, the extrapolated pressure intercept of the Ppa-Q plots approximated Pla. Oleic acid administration resulted in a parallel shift of the Ppa-Q plots to higher pressure; i.e., the pressure intercept increased, whereas the slope was not modified. Increasing Pla at constant Q before oleic acid led to a proportional augmentation of Ppa. After oleic acid, however, changes in Pla over the same range affected Ppa only at the highest levels of Pla. These results suggest that oleic acid lung injury increases the critical closing pressure that exceeds Pla, becomes the effective outflow pressure of the pulmonary circulation, and is responsible for the pulmonary hypertension.     

Nifedipine-sensitive vascular reactivity of femoral arteries in WKY: the effects of pertussis toxin pretreatment and endothelium removal

Maintenance of norepinephrine (NE)-induced contraction is dependent on Ca(2+) influx through L-type voltage-dependent Ca(2+) channels (VDCC), which is opposed by nitric oxide. Adrenergic receptors are coupled with different G proteins, including inhibitory G proteins (Gi) that can be inactivated by pertussis toxin (PTX). Our study was aimed to investigate the effects of endothelium removal, PTX pretreatment and acute VDCC blockade by nifedipine on the contractions of femoral arteries stimulated by norepinephrine. We used 12-week-old male WKY, half of the rats being injected with PTX (10 microg/kg i.v., 48 h before the experiment), which considerably reduced their blood pressure (BP). Contractions of isolated arteries were measured using Mulvany-Halpern myograph. NE dose-response curves determined in femoral arteries from PTX-treated WKY rats were shifted to the right compared to those from control WKY. On the contrary, removal of endothelium augmented NE dose-response curves shifting them to the left. Acute VDCC blockade by nifedipine (10(-7) M) abolished all differences in NE dose-response curves which were dependent on the presence of either intact endothelium or functional Gi proteins because all NE dose-response curves were identical to the curve seen in vessels with intact endothelium from PTX-treated animals. We can conclude that BP reduction after PTX injection is accompanied by the attenuation of NE-induced contraction of femoral arteries irrespective of endothelium presence. Moreover, our data indicate that both vasodilator action of endothelium and Gi-dependent vasoconstrictor effect of norepinephrine operate via the control of Ca(2+) influx through VDCC.     

Intact endothelial and smooth muscle function in small resistance arteries after 48 h in vessel culture

Long-term culture of resistance vessels allows introduction of molecular biology techniques for use in microvascular research. The aim of the present study was to establish a culture protocol that preserved vascular integrity and function in microvessels for 48 h in culture. Skeletal muscle resistance arteries were excised from the hamster gracilis muscle. Segments were assigned to immediate functional tests or to vessel culture, during which segments were perfused and superfused at a transmural pressure of 45 mmHg with Leibovitz (L15) medium containing 15% fetal calf serum and antibiotics for 48 h. Cultured and freshly isolated vessels showed similar levels of spontaneous tone, myogenic responses, changes in smooth muscle intracellular calcium (Ca(i)(2+)) (fura 2), and vascular diameter (video microscopy) in response to 0.3 M norepinephrine and similar concentration-response curves for acetylcholine (endothelium dependent, +/-N(omega)-nitro-L-arginine) and sodium nitroprusside (endothelium independent). Measurements of endothelial Ca(i)(2+) revealed similar acetylcholine-induced increases in endothelial Ca(i)(2+) in both groups. It is concluded that vascular function can be preserved while maintaining vessels in culture. Thus it is possible to utilize protocols that require long-term treatment.