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Biomedical subjects

B R Duling

Publications and source records attributed to B R Duling.

At least 37 records · Page 2Linked to original sources
PubMed1997

Acetylcholine induces conducted vasodilation by nitric oxide-dependent and -independent mechanisms.

Conducted vasodilation has been proposed as an important component of local vascular control. Because conducted vasomotor responses have previously been studied only in response to short pulses (<500 ms) of agonist, this study examined conducted vasodilation in response to sustained stimuli. In addition, we examined the contribution of nitric oxide (NO) to initiation and maintenance of conducted responses induced by acetylcholine (ACh). Responses to 2-min applications of ACh, sodium nitroprusside, and 8-bromoguanosine 3',5'-cyclic monophosphate were obtained in cannulated, perfused hamster cheek pouch arterioles (approximately 60 microm in diameter). Changes of luminal diameter in response to pressure ejection of agonists from a micropipette placed close to the downstream end of the vessel were observed at the site of stimulation ("local") as well as 570 and 1,140 microm upstream. At the local site, ACh stimuli produced large changes in diameter (approximately 70% of the maximum response) that peaked within 45 s before declining slowly to levels of approximately 50% of the maximum response. A similar response pattern was observed at both upstream sites, with the conducted responses being maintained for the duration of the stimulus. Local responses of similar magnitude were found with sodium nitroprusside and 8-bromoguanosine 3',5'-cyclic monophosphate, but only minimal responses were observed at the conducted sites. In a separate set of arterioles, ACh responses were obtained before and during perfusion with 10 microM N(omega)-nitro-L-arginine. Inhibition of NO synthesis diminished the local response to ACh, but the initial phase of the conducted response was unaffected. Furthermore, the conducted responses faded more rapidly in the presence of N(omega)-nitro-L-arginine. We conclude from these results that local NO synthesis alone is insufficient to initiate conducted responses but that NO synthesis contributes to maintenance of sustained conducted responses.

Acetylcholine↗
PubMed1996

Inosine-induced vasoconstriction is mediated by histamine and thromboxane derived from mast cells.

Mast cell degranulation has been shown to release products that cause arteriolar constriction. We previously reported that two nucleosides, adenosine and inosine, cause vasoconstriction of isolated hamster cheek pouch arterioles by stimulating degranulation of periarteriolar mast cells. The objectives of the present study were to characterize the nucleoside-dependent vasoconstriction in vivo and to determine the mediator or mediators responsible. We examined the vasomotor effect of inosine on arterioles in the cheek pouches of anesthetized hamsters (70 mg/kg pentobarbital sodium) in the control situation and in the presence of receptor antagonists for histamine (H1), thromboxane A2 (Tx), and leukotrienes (LT). Most experiments were carried out using inosine applied once locally via micropipette to arterioles and observing the subsequent response. Over a range of inosine concentrations from 10(-5) to 10(-3) M in the pipette, we observed a dose-dependent increase in the incidence and magnitude of constriction. In addition, mast cell staining with ruthenium red was observed after stimulation with inosine, an indication of mast cell degranulation. Neither the H1, Tx, nor LT antagonist alone had a significant effect on the vasomotor response to inosine. However, combined H1 and Tx blockade significantly reduced the incidence and magnitude of inosine-induced constriction. These data establish that inosine-induced constriction occurs in vivo and support the role of mast cells in this response. Furthermore they suggest that multiple mediators, primarily histamine and thromboxane, are responsible for the observed constriction.

Animals↗
PubMed1996

Ratiometric measurement of endothelial depolarization in arterioles with a potential-sensitive dye.

A fluorescence ratio technique based on the voltage-sensitive dye 1-(3-sulfonatopropyl)-8-[beta-[2-di-n-butylamino)-6-naphythyl++ +]vinyl] pyridinium betaine (di-8-ANEPPS)has been developed for recording membrane potential changes during vascular responses of arterioles. Perfusion of hamster cheek pouch arterioles with the dye labeled the endothelial cell layer. voltage responses from the endothelium of intact arterioles were determined by analysis of voltage-induced shifts in fluorescence emission wavelengths from dye spectra imaged from the vessel wall. Membrane depolarization caused the dye spectrum to shift toward blue wavelengths, with maximal fluorescence changes near 560 and 620 nm. In isolated nonperfused arterioles, comparison of continuous dual-wavelength recordings with simultaneous microelectrode recordings showed that the ratio of fluorescence intensities (fluorescence at 620 nm to fluorescence at 560 nm) accurately followed changes in membrane potential (6-21 mV) during vasoconstriction. The dye response was linear with respect to potential changes from -56 to -6 mV, with a voltage sensitivity of 9.7% change in the ratio per 100 mV. Membrane potential responses from in vitro and in vivo arterioles after potassium stimulation consisted of rapid ( < 0.5 -s) depolarization followed by slow repolarization over several seconds. Potassium-induced depolarizations were conducted along arterioles, and the values of the electrical length constant for conducted depolarization determined by optical and microelectrode methods were in agreement. We conclude that ratio analysis of di-8-ANEPPS fluorescence emission can be used to accurately record membrane potential changes on the time scale of seconds during vasomotor activity from arterioles.

Animals↗
PubMed1996

Adenosine-induced vasoconstriction in vivo. Role of the mast cell and A3 adenosine receptor.

Adenosine, a vasodilator metabolite, is often produced in tissues where the demand for oxygen exceeds the supply. We have recently demonstrated in isolated cannulated arterioles that adenosine and its metabolite, inosine, can also cause vasoconstriction by stimulation of mast cells. Secondary release of histamine and thromboxane is responsible for the inosine-induced constriction in vivo. In the present study, we explored the vasomotor effects of adenosine in vivo and investigated the role of the A3 adenosine receptor in mediating vasoconstriction. In vivo, local application of adenosine (10-6 to 10-4 mol/L) to arterioles consistently caused dose-dependent vasodilation. A fraction of arterioles, however, exhibited a biphasic response, with constriction following dilation. This, too, was dose dependent; 37% of arterioles constricted by 12.7 +/- 4.3% of the initial diameter in response to 10-4 mol/L adenosine. In the presence of 8-(p-sulfophenyl)theophylline (8-SPT), an antagonist of A1 and A2 adenosine receptors, dilation in response to the same dose of adenosine was reduced, and constriction was enhanced; 85% of the tested arterioles constricted by -44.3 +/- 6.0% of the initial diameter. The A3 adenosine receptor has been shown to facilitate mediator release from mast cells, and its role was also examined. N6-(3-Iodo-4-aminobenzyl)adenosine (I-ABA), an agonist of A1 and A3 adenosine receptors, produced dose-dependent vasoconstriction. 1,3-Dipropyl-8-(4-acrylate)phenylxanthine (BW-A1433), an antagonist of A1, A2, and A3 receptors, significantly reduced the vasoconstrictor response to adenosine, which was unmasked during treatment with 8-SPT. In addition, both adenosine and I-ABA stimulated mast cell uptake of ruthenium red, indicating degranulation. The I-ABA-induced constriction was abolished by combined histamine and thromboxane receptor antagonists. We conclude that adenosine can cause vasoconstriction in vivo, which is often masked by A2 receptor-mediated vasodilation. Mast cells are stimulated in the course of the response, and the A3 adenosine receptor is involved in mediating constriction.

Adenosine↗
PubMed1996

Identification of distinct luminal domains for macromolecules, erythrocytes, and leukocytes within mammalian capillaries.

A thick endothelial surface coat consisting of the glycocalyx and associated plasma proteins has been hypothesized to reduce functional capillary volume available for flowing plasma macromolecules and blood cells. The purpose of this study was to compare anatomic and functional capillary diameters available for macromolecules, RBCs, and WBCs in hamster cremaster muscle capillaries. Bright-field and fluorescence microscopy provided similar estimates (mean +/- SE) of the anatomic capillary diameter: 5.1 +/- 0.1 microns (bright field, 39 capillaries in 10 animals) and 5.1 +/- 0.2 microns (membrane dye PKH26, 18 capillaries in 2 animals). Estimates of functional diameters were obtained by measuring the width of RBCs and WBCs and the intracapillary distribution of systemically injected fluorescein isothiocyanate (FITC)-dextran 70. WBCs (5.1 +/- 0.2 microns) fully occupied the anatomic capillary cross section. In contrast, the widths of RBCs (3.9 +/- 0.2 microns, 21 capillaries in 8 animals) and FITC-dextran (4.3 +/- 0.2 microns, 21 capillaries in 8 animals) were significantly smaller than the anatomic capillary diameter. Continuous (1- to 5-minute) excitation of fluorochromes in the capillary lumen (light-dye treatment) increased the width of RBCs passing the treated site from 3.6 +/- 0.3 to 4.4 +/- 0.3 microns (6 capillaries in 4 animals) and the width of the FITC-dextran column from 4.1 +/- 0.2 to 4.6 +/- 0.3 microns (10 capillaries in 7 animals). Furthermore, light-dye treatment increased capillary tube hematocrit by 60% in 40-microns-long capillary segments compared with untreated sites in the same capillaries. It is concluded that the wall of skeletal muscle capillaries is decorated with a 0.4- to 0.5-microns-thick endothelial surface coat, which may represent the true active interface between blood and the capillary wall.

Animals↗
PubMed1995

Method to create small photo-bleached volumes to monitor blood plasma flow in capillaries.

A method has been developed to examine the movement of plasma in capillaries using intravital microscopy. Spatial transients in fluorescence properties are instantaneously induced by laser photo-bleach pulses after which the convective recovery can be monitored. The plasma is tagged with fluorescent dyes coupled to bovine serum albumin, which is injected well before the measurements and circulates with the blood stream. A laser beam from an argon laser source, set to emit light with a wavelength of 488 nm, is focused on the illumination field diaphragm and creates a spot in the object plane of the microscope. At low laser power, the laser spot is aimed at a blood plasma gap between red blood cells in a capillary segment, using a steerable mirror. Light sensors, coupled to photo-multipliers in the secondary image plane of the microscope, record the light intensity of the moving plasma/dye while the preparation is continuously illuminated with a xenon epi-illuminating set-up. The laser photo-bleach spot is then used to bleach the dye complex within a 5.4 microns segment of the capillary for less than 20 ms. The movement of the bleached plasma bolus is tracked by the photo-sensors, placed sequentially along the capillary. Both dye and red blood cell passage can be detected in the photo-multiplier signals, and the relative velocities of the two blood components can be measured. Measurements reveal that the ratio of transit times between blood plasma and red blood cells is 1.23 (SD = 0.22, N = 18), which is in good agreement with measurements by other techniques.

Animals↗
PubMed1995

Connexin 43 and connexin 40 gap junctional proteins are present in arteriolar smooth muscle and endothelium in vivo.

The distributions of connexin 43 (Cx43) and connexin 40 (Cx40) in smooth muscle and endothelium of resistance vessels were examined using indirect immunofluorescence techniques coupled with confocal microscopy. Cx43 and Cx40 were found in smooth muscle and endothelium. Similar staining patterns were found in microvessel samples from brain and cremaster of the rat and from arterioles of the hamster cheek pouch. Double-labeling studies showed a high degree of colocalization of Cx40 with Cx43, suggesting the presence of multiple connexins within a single junctional plaque. Quantitative comparisons were made of the fluorescent patterns in the endothelium and smooth muscle of rat brain arterioles. Cx43 and Cx40 plaque diameters were 0.9 +/- 0.1 and 0.8 +/- 0.1 (SE) microns, respectively, in the endothelial layer and 0.5 +/- 0.1 and 0.5 +/- 0.1 microns, respectively, in the smooth muscle. There was no difference between mean plaque diameters of Cx43 and Cx40 in endothelium or smooth muscle. However, plaques were significantly larger in endothelium than in smooth muscle (P < 0.05). These findings demonstrate the potential for cell-cell communication in both cell types of the wall of arterioles from three different tissues. The data also suggest a greater level of coupling within the endothelium.

Animals↗
PubMed1995

Electromechanical coupling and the conducted vasomotor response.

Conducted vasomotor responses are viewed as one mechanism that functionally integrates the microvasculature. It is hypothesized that the conducted vasomotor response is the result of an electrical current and its passive electrotonic spread along the length of a microvessel. We tested this hypothesis in isolated, unpressurized arterioles from the hamster cheek pouch using conventional intracellular membrane potential recording techniques. The mean resting membrane potential (RMP) was -67 mV. KCl and phenylephrine (PE) pulse-stimulation applied through micropipettes could both induce transient depolarizations and vasoconstrictions at the site of stimulation (local) and at conducted (560 microns) sites. It was noted, however, that the conducted vasomotor response could not be induced until the conducted electrical response exceeded a threshold of -45 mV for a minimum amount of time. The relationship between the amplitude of constriction and the amplitude-time area of depolarization above -45 mV was the same for local and conducted KCl and for conducted PE but was significantly different from that for local PE. Nifedipine greatly reduced the local and conducted mechanical but not electrical responses. Our results indicate that the conducted vasomotor responses are the result of the generation and subsequent conduction of electrical signals along the vessel but that the corresponding mechanical response occurs only when the electrical response exceeds a threshold level.

Animals↗
PubMed1995

Cellular pathways of the conducted electrical response in arterioles of hamster cheek pouch in vitro.

We have previously shown that conducted vasomotor responses follow patterns that are consistent with a passive spread of electrical current along the length of the arterioles [(Xia and Duling, Am. J. Physiol. 269 (Heart Circ. Physiol. 38): H2022-H2030, 1995]. In this study, we define the cells through which the current flows. Isolated arterioles of hamster cheek pouch were used. The mean resting membrane potential (RMP) for randomly sampled arteriolar cells was -67 mV. When cell types were identified by dye injection, the RMPs were -68 and -67 mV for smooth muscle (SM) and endothelium (EC), respectively. Pulses of KCl induced transient, monophasic depolarizations at the site of stimulation (local), which were conducted decrementally along the length of the arteriole over several millimeters. During electrical conduction, three patterns of responses could be observed, but identical patterns of the conducted electrical responses were always observed in SM and EC. Phenylephrine stimulation also caused transient local and conducted depolarizations in both SM and EC. As with KCl stimuli, shapes of conducted electrical responses were identical in records made in both cell types. The results suggest that SM and EC are electrically coupled both homocellularly and heterocellularly.

Alcohols↗
PubMed1995

Dye tracers define differential endothelial and smooth muscle coupling patterns within the arteriolar wall.

Dye tracers were chosen, based on net charge, chemical structure, and reactive groups, to test for the existence of and to provide novel insight into channel selectivities of junctional pathways connecting smooth muscle and endothelial cells of the arteriolar wall. Dyes were injected into individual smooth muscle or endothelial cells of hamster cheek pouch arterioles using microiontophoresis. Coupling, independent of tracer net charge, was seen both within and between cell layers. Endothelial cells were well coupled by all of the tested dyes. Smooth muscle junctions appeared less effective in dye transfer than endothelial junctions. Lucifer yellow was confirmed to be a poor tracer of smooth muscle gap junctions, and remarkably this dye and other related sulfate-containing molecules interfered with dye movement through smooth muscle but not endothelial junctions. Myoendothelial junctions showed a striking polarity of dye movement, with dye transfer from endothelial to smooth muscle cells but little or no transfer in the reverse direction. Because the dyes have size and charge characteristics similar to those of known cellular second messengers, these findings have important implications for cell-cell signaling in the vessel wall.

Animals↗
PubMed1995

Use of Ruthenium Red staining to detect mast cell degranulation in vivo.

OBJECTIVE: To establish a method of detecting mast cell degranulation in tissues during in vivo microscopy. METHODS: Hamster tissues were prepared for intravital microscopy. Ruthenium red (RR) was superfused over the cheek pouch at concentrations of 0.0001-0.01% to determine the optimal concentration. Mast cells were stimulated with compound 48/80, as well as with vasoactive agents not known to be stimulatory to mast cells, following which, mast cell staining was observed. Mesenteries were stained with Toluidine Blue (TB) or RR and mast cell degranulation was assessed during treatment with compound 48/80, or control. RESULTS: During superfusion with varying concentrations of RR, a dose dependence for background staining of unstimulated cells was observed. A RR concentration of 0.001% was optimal for in vivo detection of mast cell degranulation. Mast cells exposed to 0.001% RR were stained following stimulation with compound 48/80 but not after treatment with KCl or acetylcholine. The latter agents are not known to stimulate mast cells. Thus, arteriolar vasomotor responses, per se, did not appear to play a role in mast cell RR uptake. Comparable results were obtained with RR versus TB in control or 48/80-treated mesenteries. CONCLUSIONS: This RR technique facilitates rapid detection of mast cell degranulation in vivo and provides an opportunity to assess both mast cell and microvascular function simultaneously.

Animals↗
PubMed1994

Nucleoside-induced arteriolar constriction: a mast cell-dependent response.

Adenosine (Ado) is a potent vasodilator that has occasionally been shown to cause vasoconstriction. Constrictor responses are generally attributed to A1-receptor stimulation or interactions with the renin-angiotensin system. We describe a previously unreported vasoconstrictor action of Ado and inosine (Ino) in hamster cheek pouch arterioles and examine the mechanism by which these nucleosides induce constriction. Arterioles were dissected from male Golden hamster cheek pouches, transferred to a 37 degrees C tissue chamber, and cannulated at both ends. Changes of luminal diameter in response to Ado were measured to generate cumulative concentration-response curves. The concentration-response curves were biphasic: 10(-6) M Ado elicited an intense, transient constriction, and higher concentrations induced dilator responses. Pretreatment with 8(p-sulfophenyl)theophylline, an Ado receptor antagonist, inhibited the dilator responses but did not alter the constriction. Inhibition of Ado uptake with S-(4-nitrobenzyl)-6-thio-inosine eliminated the constrictor response without altering dilator responses. Similar effects were found after pretreatment with an Ado deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride. Finally, Ino, a metabolite of Ado, induced constrictions of similar magnitude to those seen with Ado, but at higher concentrations. The constrictor response was focal in nature, suggesting discrete sites of action of Ado. Methylene blue staining after Ado application revealed degranulated mast cells closely associated with the vessel wall, indicating a possible role for mast cell degranulation in the constrictor response. Supporting this idea were the observations that inhibition of degranulation by 10 microM cromolyn blocked the constrictor response, and compound 48/80 (a mast cell secretagogue) caused constriction similar to that elicited by Ado.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine↗
PubMed1994

Determination of capillary tube hematocrit during arteriolar microperfusion.

Intracapillary hematocrit is known to be substantially lower than arterial hematocrit. We hypothesized that capillary hematocrit might be influenced by interactions between plasma macromolecules and the endothelial cell surface. Microvessel perfusion pipettes were inserted in second- or third-order vessels, and capillaries were perfused with three different artificial bloods composed of 50% red cells plus the following suspension media: fetal calf serum (group I), serum albumin plus serum globulins (fractions II and III; group II), and bovine serum albumin plus dextran (group III). The mean hematocrits of the pipette-perfused capillaries averaged close to 50% of the systemic value with all perfusion fluids and were not different from the hematocrits of the capillaries perfused by the animal. These data suggest that bifurcations proximal to the pipette location did not contribute to the reduction in mean tube hematocrit normally seen in the animal. Furthermore, interactions between the plasma macromolecules and the endothelial cell surface do not appear to contribute to the low intracapillary hematocrit. Analysis of the data indicate that the capillary Fåhraeus effect, the network Fåhraeus effect in terminal vessels of the arterial tree, and intracapillary events all contribute to the reduction in intracapillary hematocrit.

Adenosine↗
PubMed1994

Vulnerability of conducted vasomotor response to ischemia.

Many vasoactive substances induce two responses, a direct effect at the site of application and a conducted response that spreads along the vessel length. In the microcirculation, we find that these two components of the vasomotor response display quite different sensitivities to occlusion and/or ischemia. Conducted vasomotor responses were induced in arterioles of the hamster cheek pouch by micropipette application of two test agents: phenylephrine (PE), which causes a receptor-mediated vasomotor response, and KCl, which causes an alteration in the membrane potential by a simple change in the K+ gradient. Ischemia was produced either by total occlusion of the vascular supply, which resulted in a complete cessation of flow in all vessels, or by venous occlusion, which was achieved by gradually inflating a pressurized cuff positioned across the pedicle of the pouch until venous return from the pouch was arrested while the feed arterioles remained patent. Both types of occlusion produced ischemia, the former with low intravascular pressure, the latter with high intravascular pressure. During both types of occlusion, arterioles were initially maximally dilated and unresponsive to both agonists, but over a subsequent 3- to 5-min period, resting arteriolar tone and local responses to both agonists returned. With total occlusion, the conducted response to KCl returned in parallel with the local response, whereas the conducted response to PE was diminished or absent. With venous occlusion, the local responses recovered as with total occlusion, but the conducted responses to both PE and KCl recovered as well.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

A micropipette which allows in situ perfusion of arterioles and capillaries.

In order to investigate capillary physiology, a glass micropipette system was developed that allowed in situ perfusion of microvessels as well as rapid changes of perfusion solutions. Theta tube (WPI, Inc.; 1.5-mm o.d. glass stock capillary tubing which is divided into two hemicylindrical sides by a central glass septum) was pulled to a smaller diameter of approx 300-600 microns and inserted into the shank of a sharpened cannulating micropipette tip constructed from large-bore glass stock (1.6 mm i.d.). The resulting dead volume between the end of the Theta supply tube and the tip of the outer cannulating tip was approximately 90 nl. The perfusate was driven in a circuit from a pressurized feed reservoir down one side of the Theta supply tube pipette and back through the second side into a reservoir maintained at a lower pressure. The pressure gradient between the two reservoirs established a high-volume flow rate and subsequently a short perfusate transit time from the feed to the collection reservoir. The average pressure in the two reservoirs determined the pressure which drove the perfusate from the cannulating tip. At normal pressures and flows, the time required to change perfusion fluid composition at the pipette tip was less than 1 min, and discharge hematocrit of a red blood cell suspension was indistinguishable from the hematocrit measured in the feed reservoir.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

A technique for the estimation of plasma flow in single capillaries using photobleached dyes.

In order to estimate plasma flow in single capillaries, an "indicator bolus" was optically inserted into individual microvessels of the hamster cremaster muscle. This was accomplished using short-duration (200 msec), argon laser pulses to photobleach a 5-microns segment of fluorochrome circulating with the plasma. The subsequent motion of the bleached plasma bolus was then tracked using photomultipliers positioned at three sites along the capillary. The transient passage of the dye appeared as a steep fall in light intensity as the downstream edge of the bleached area entered the sensor field, followed by a steep rise in light intensity as trailing unbleached plasma flowed under the sensor. The behavior of light intensity as the photobleached bolus flowed past a sensor was analyzed using a theoretical model developed to predict the behavior of this type of plasma flow indicator in single capillaries. The characteristic time, tau, which equals the capillary segment volume divided by the plasma flow, was taken as an estimate of plasma flow. The model predicts that, for this system, tau of the capillary corresponds closely to the time at which 50% of the full sensor response to the bolus is attained, that is, the t50. The ratio between the t50 and the characteristic time is found to be a function of the relative sensor width and the flow velocity profile. A procedure is also described to assess the flow velocity profile from in vivo measurements. Using this technique, the ratio of the velocity of the red cell compared to that of plasma is found to be about 1.3.

Animals↗