Microvessels Of Mesentery Research Articles

Ca2+ entry through conductive pathway modulates receptor-mediated increase in microvessel permeability.

We investigated the relationship between receptor-mediated increases in cytoplasmic Ca2+ concentration ([Ca2+]i) and increased microvessel permeability. In individually perfused venular microvessels of frog mesentery exposed to 10 microM ATP, [Ca2+]i increased from 59 +/- 7 to 172 +/- 21 nM within 1 min and then fell back toward control values. The corresponding peak increase in the hydraulic conductivity (Lp) of the microvessel wall was 5.7 +/- 0.5-fold relative to control. After removal of extracellular Ca2+, there was no significant increase in Lp, and the initial increase in [Ca2+]i was attenuated but not abolished. Depolarization of the endothelial cell membrane with high-K+ Ringer solution reduced the peak increase in [Ca2+]i to 106 +/- 7 nM and attenuated the increase in Lp 1.8 +/- 0.4-fold. The results conform to the hypothesis that Ca2+ entry into endothelial cells is required for acute increase in venular microvessel permeability by inflammatory agents and that the pathway for Ca2+ entry has the properties of a passive conductance pathway. Similar conclusions were reached in previous experiments in frog microvessels exposed to Ca2+ ionophores and perfusates with no plasma proteins. In venular microvessels of hamster mesentery exposed to ATP and bradykinin, a similar pathway for Ca2+ entry was demonstrated in the present experiments. We did not measure permeability changes in hamster microvessels in this study, but these microvessels respond to histamine and ionophores with a transient increase in permeability to macromolecules similar to that measured in frog microvessels [Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H1982-H1991, 1995].

Solvent drag of LDL across mammalian endothelial barriers with increased permeability.

We investigated the mechanisms of hamster low-density lipoprotein (LDL) transport across the endothelial barrier in individually perfused venular microvessels in hamster mesentery. These experiments are the first to use microperfusion techniques and quantitative fluorescence microscopy to investigate LDL transport across mammalian microvessel endothelium. The apparent permeability coefficient for hamster LDL, PsLDL, rose from 2.7 x 10(-7) cm/s at control to 23.2 x 10(-7) cm/s at the peak of the biphasic increase in microvessel permeability after exposure of the vessels to 100 microM histamine. Close to the peak, PsLDL rose 1.85 x 10(-7) cm/s for every centimeter of H2O increase in hydrostatic pressure. Thus, at a mean pressure of 11.3 cmH2O, 90% of the LDL flux was coupled to transendothelial water flow by a solvent drag mechanism. The corresponding solvent drag reflection coefficient for hamster LDL was estimated to be approximately 0.8. These results are consistent with sieving hamster LDL (effective radius 14.9 nm) through equivalent pores of approximately 22 nm radius. Similar results were found with human LDL (effective radius 13.2 nm) in hamster microvessels. The results provide a bridge between studies of LDL transport across cultured endothelial barriers, where high diffusive permeability coefficients to LDL may obscure the contributions of solvent drag, and studies in whole animals, where the consequences of sieving of LDL at the vessel wall, even in the high permeability state, have not received much attention.

In vivo evidence for microvascular oxidative stress in spontaneously hypertensive rats. Hydroethidine microfluorography.

The factors that predispose to the accelerated organ injury that accompanies the hypertensive syndrome have remained speculative and without a firm experimental basis. Indirect evidence has suggested that a key feature may be related to an enhanced oxygen radical production. The purpose of this study was to refine and use a technique to visualize evidence of spontaneous microvascular oxidative stress in vivo in the spontaneously hypertensive rat (SHR) compared with its normotensive control, the Wistar-Kyoto rat (WKY). We investigated the effects of adrenal glucocorticoids on the microvascular oxidative stress sequence. The mesentery was superfused with hydroethidine, a reduced, nonfluorescent precursor of ethidium bromide. In the presence of oxidative challenge, hydroethidine is transformed intracellularly into the fluorescent compound ethidium bromide, which binds to DNA and can be detected by virtue of its red fluorescence. The fluorescent light emission from freshly exteriorized and otherwise unstimulated mesentery microvessels was recorded by digital microscopy. The number of ethidium bromide-positive nuclei along the arteriolar and venular walls in SHR was found to be significantly increased above the level exhibited by WKY. The elevation in ethidium bromide fluorescence in SHR arterioles could be attenuated by a synthetic glucocorticoid inhibitor and in rats subjected to adrenalectomy. The administration of glucocorticoids after adrenalectomy by injection of dexamethasone restored the oxidative reaction in SHR arterioles. Treatment with dimethylthiourea and with a xanthine oxidase inhibitor attenuated the superoxide formation. Although a nitric oxide synthase inhibitor (NG-nitro-L-arginine methyl ester) enhanced the ethidium bromide staining in WKY, it did not affect that in SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Imaging of Ca2+ transients in endothelial cells of single perfused capillaries: correlation of peak [Ca2+]i with sites of macromolecule leakage.

To investigate the mechanisms responsible for variation in the macromolecular leakage (formation of localized leaky sites) in venular microvessels with increased permeability, we examined the hypothesis that cytoplasmic calcium concentration [Ca2+]i, does not increase uniformly within microvessel endothelial cells. We loaded the endothelial cells forming the walls of venular microvessels in frog mesentery with fura-2, and imaged [Ca2+]i using a cooled CCD camera. Control [Ca2+]i was close to 60 nM in all regions. Control permeability was uniformly low in all microvessels. Exposure to ionomycin (5 mM) increased [Ca2+]i in a biphasic manner, but not uniformly. There was variation in both time to peak (bimodal distribution) and peak [Ca2+]i (274 +/- 13 nM; mean variation above or below the peak value was 110 nM). Raising extracellular calcium from 1.1 to 5 mM increased the mean variation of [Ca2+]i about peak values. Extravascular leakage of fluorescently labeled albumin or low-density lipoproteins was most prominent at sites where increase in [Ca2+]i were largest. These data indicate that variation in [Ca2+]i within individual endothelial cells or groups of cells could account, at least in part, for the distribution of localized leakage sites for macromolecules in venular microvessels in the high-permeability state.

Microscopic laser Doppler velocimeter measuring blood velocity in single microvessel.

A microscopic laser Doppler velocimeter to measure blood velocity in single microvessels was developed. In order to measure the blood velocity in microvessels correctly, the influence of stray scattered light from vessel wall should be removed. Because it provokes low-frequency spurious components. The spectra of the spurious components overlap with that of Doppler signal and induces considerable error in the result of the blood velocity when the velocity is reduced. The optical configuration of the velocimeter was optimized to eliminate the influence of the stray scattered light. The blood velocity in microvessels in rat mesentery was measured with the microscopic laser Doppler velocimeter and the periodical fluctuation of the velocity was analyzed. Pulsatile velocity fluctuation could be observed even in capillary.

Measurement of cytoplasmic calcium in single microvessels with increased permeability

We investigated the hypothesis that an increase in cytoplasmic calcium ion concentration, [Ca2+]i, is one of the mechanisms responsible for increased microvessel permeability. We loaded the cells forming the walls of individually perfused microvessels in frog mesentery with fura-2 and measured [Ca2+]i in the control state and after adding the Ca2+ ionophore ionomycin to the perfusate. [Ca2+]i in the control state was 65 +/- 6 nM and increased to an initial peak of 285 +/- 29 nM after 1-3 min. After 4, 6, and 10 min, [Ca2+]i was 199 +/- 18, 163 +/- 16, and 129 +/- 9 nM, respectively. [Ca2+]i fell back to 77 +/- 7 nM after ionophore was removed. In similar experiments, hydraulic conductivity (Lp) increased to a peak of 9.5 times control after 1-3 min, then fell to 2.0 times control after 6 min. Lp remained elevated at this level for as long as ionophore was present in the perfusate. [Ca2+]i modulates the initial and sustained phases of the permeability increase. Both processes depend on external Ca2+ influx. Our experiments provide the first direct measurement of [Ca2+]i during a change in the permeability of an intact microvessel.

Modulation of microvessel wall charge by plasma glycoprotein orosomucoid

Haraldsson and Rippe suggested that the circulating glycoprotein orosomucoid (alpha 1-acid glycoprotein) contributes to the net charge on microvessel walls (Acta Physiol. Scand. 129: 127-135, 1987). We tested their hypothesis in individually perfused microvessels of frog mesentery by measuring solute permeability coefficients of two globular proteins (alpha-lactalbumin and ribonuclease) having approximately the same size (Stokes radius, 2 nm) but different charge (-11 and +3, respectively). In vessels perfused with orosomucoid (0.1 and 1 mg/ml) in a Ringer-albumin perfusate, the solute permeability coefficient of alpha-lactalbumin decreased to one-half [0.47 +/- 0.25 (SD)] the value in the absence of orosomucoid, and the solute permeability coefficient of ribonuclease was close to six times as large as alpha-lactalbumin permeability. Both results may be accounted for if orosomucoid increases the net negative charge on microvessel walls in frog mesentery from 11.2 to 28 meq/l. A similar change in microvessel charge would be more than sufficient to account for the decrease in albumin clearance in the presence of orosomucoid reported by Haraldsson and Rippe in rat muscle microvessels.

Effect of physical training on thrombotic tendency in rats: decrease in thrombotic tendency measured by the He-Ne laser-induced thrombus formation method.

The effect of physical training on thrombotic tendency was assessed in rats. Exercise was done on a flat treadmill for 30 min at a rate of 1,400 m/h (submaximal speed), 5 times a week for either 1.5 or 3 months. The thrombotic tendency was measured by the He-Ne laser-induced thrombus formation method in microvessels of mesentery, i.e. measurement of the number of laser irradiations necessary to induce stasis of blood flow by occlusive thrombus formation. An increase in the number of irradiations necessary to induce occlusive thrombus formation was observed in arterioles, but not in venules after physical training for 1.5 and 3 months.

Single capillary permeability to proteins having similar size but different charge.

We investigated the hypothesis that solute charge modulates transcapillary exchange in microvessels with continuous endothelium. Two globular proteins, alpha-lactalbumin and ribonuclease, having approximately the same size (mol wt 14,176 and 13,683, respectively) but different net charge (-10 and +4, respectively) were test solutes. Each solute was labeled with the fluorescent probe tetramethylrhodamine isothiocyanate. Labeling did not significantly change solute size, but increased negative charge on each solute by one valency unit. An in vivo fluorescent microscope technique [Huxley et. al., Am. J. Physiol. 252 (Heart Circ. Physiol. 21): H188-H197, 1987] was used to measure solute permeability coefficients (P) in single microvessels of frog mesentery at 14-16 degrees C. The mean P for alpha-lactalbumin, measured when capillary pressure was 10 cmH2O, was 2.1 X 10(-6) cm/s and the mean P for ribonuclease was 4.3 X 10(-6) cm/s. Our results conform to the hypothesis that the transcapillary pathways of frog mesenteric microvessels are negatively charged. With the use of a Donnan-type model for electrostatic partitioning, charge density in the pathway is estimated as 11.4 meq/l. Comparison of measured Ps with those for small solutes in frog mesenteric microvessels indicates that molecular size is a proportionally more significant determinant of solute permeability in continuous capillaries than is solute charge.

Effect of pentoxifylline on microvascular blood flow velocity.

Pentoxifylline (PF), known to increase red blood cell (RBC) deformability, has recently attracted much attention because of its possible effect of reducing capillary resistance. There has been practically no reliable demonstration of an increase in capillary blood flow by this drug. This study was intended to examine the effect of PF and hence deformability changes on microvascular hemodynamics and the rheological mechanisms underlying such an effect. Mesentery microvessels of 15 rats were subjected to intravital microscopic observation under transillumination. Capillary blood flow was measured using a newly developed 2-channel sample-hold scanner incorporated into a video display system. Changes in whole blood viscosity were also measured in order to investigate hemorheological changes. It was found that a significant increase in capillary blood flow (as much as 140%) was brought about by PF administration. The increase in blood flow was particularly remarkable in capillaries of about 13 microns in diameter, while whole blood viscosity measured in vitro showed a reduction of 40% at most. In view of these results and the reported effect of PF on increasing the RBC deformability, increased capillary blood flow can be attributed to the increased RBC deformability.

Dispersion of indicator measured from microvessels of cat mesentery.

The indicator-dilution method was used to investigate blood flow in the microvascular network of the mesenteric membrane of the cat by replacing the normal blood flow into the mesenteric artery with a dextran-saline solution for a finite period of time. The dilution of the dextran-saline bolus as it flowed downstream was measured in a selected arteriole and its adjacent venule with a microphotometric system. Based on an in vitro calibration study done on glass capillary tubes, the measured optical density was converted to the hematocrit. The dilution curve for a finite injection and for a step injection, the mean transit time (MTT), and the appearance time for the arteriole and the venule were computed. The dispersion found in the arterial system, a network of diverging branches, was reasonably well simulated by the dispersion in a parabolic flow system. However, the dispersion found in the venules, where the irregular capillary blood flow converges, was considerably skewed from that in a parabolic system. A wide distribution of MTT was found for the arterioles and the venules, and there was considerable overlap in the MTT distribution for these two groups. This finding cannot be simulated by a network with a parallel arrangement. The difference in MTT for pairs of arterioles and venules was distributed over a narrow range, probably indicating shunt flow in the mesenteric microvascular network.

Effects of intra-arterially injected contrast media on microvessels in the rat's cecal mesentery.

The effect of contrast media on the permeability of microvessels in the rat's cecal mesentery was examined in vivo. Contrast media were injected intraarterially and flowed through the vessels under observation for 30 seconds. Subsequent disposition of injected colloidal carbon particles was used as a marker for permeability changes. Microscopic observations and photographic records were made of the vessels exposed to the radiopaque materials. There was no significant increase in the permeability of normal microvessels after exposure to contrast materials. An increase in permeability due to exposure to contrast media was noted in two cases: 1) in vessels damaged during preparation for observation before exposure to contrast media, and 2) in vessels which became occluded after exposure to contrast media. The purpose of this study was to evaluate the permeability effects of intra-arterial injection of contrast materials following relatively short exposure of microvessels to these hyperosmolar solutions.

Recording of microvascular dimensions with an image-splitter television microscope.

Recording of microvascular dimensions with an image-splitter television microscope.