Theoretical model of metabolic blood flow regulation: roles of ATP release by red blood cells and conducted responses

A proposed mechanism for metabolic flow regulation involves the saturation-dependent release of ATP by red blood cells, which triggers an upstream conducted response signal and arteriolar vasodilation. To analyze this mechanism, a theoretical model is used to simulate the variation of oxygen and ATP levels along a flow pathway of seven representative segments, including two vasoactive arteriolar segments. The conducted response signal is defined by integrating the ATP concentration along the vascular pathway, assuming exponential decay of the signal in the upstream direction with a length constant of ∼1 cm. Arteriolar tone depends on the conducted metabolic signal and on local wall shear stress and wall tension. Arteriolar diameters are calculated based on vascular smooth muscle mechanics. The model predicts that conducted responses stimulated by ATP release in venules and propagated to arterioles can account for increases in perfusion in response to increased oxygen demand that are consistent with experimental findings at low to moderate oxygen consumption rates. Myogenic and shear-dependent responses are found to act in opposition to this mechanism of metabolic flow regulation.

Participation of intracellular Ca2+ stores in arteriolar conducted responses

We examined the role played by intracellular Ca2+ stores in conducted vasomotor responses induced by phenylephrine (PE) in isolated hamster cremasteric arterioles. When applied briefly (∼1 s) to isolated, cannulated arterioles by using pressure-pulse ejection from a micropipette, PE produced a strong local vasoconstriction and a very small biphasic conducted response (a small constriction followed by a dilation) that propagated several hundred micrometers along the vessel length. The conducted vasomotion was associated with a monophasic elevation of the endothelial cell intracellular Ca2+ concentration ([Ca2+]i) at the site of stimulation, as measured with the Ca2+ indicator fura 2. The Ca2+ pump inhibitor thapsigargin was used to limit filling of Ca2+ stores in smooth muscle and endothelial cells. Thapsigargin reduced baseline diameter and elicited a strong dilator component at the local site while enhancing both the constrictor and dilator components of the PE-induced conducted response. The enhanced conducted constrictor component induced by thapsigargin was mimicked by extraluminal application of tetraethylammonium or charybdotoxin but not by iberiotoxin, apamin, glibenclamide, barium, or 4-aminopirydine. Thapsigargin increased the estimated basal endothelial cell [Ca2+]i by ∼60 nM and converted the PE-induced change in [Ca2+]i from monotonic to biphasic with a late elevation of [Ca2+]i above baseline that coincided with the increased dilatory component of the conducted response. Luminal application of charybdotoxin plus apamin significantly reduced the dilatory component of the conducted response. These results indicate that intracellular Ca2+ stores play a dynamic role in regulating conducted vasomotor responses apparently through modulation of KCa channels in both cell types.

A new method for assessing arteriolar diameter and hemodynamic resistance using image analysis of vessel lumen

To characterize the nonuniform diameter response in a blood vessel after a given stimulus (e.g., arteriolar conducted response), frequent serial diameter measurements along the vessel length are required. We used an advanced image analysis algorithm (the “discrete dynamic contour”) to develop a quick, reliable method for serial luminal diameter measurements along the arteriole visualized by intravital video microscopy. With the use of digitized images of the arteriole and computer graphics, the method required an operator to mark the image of the two inner edges of the arteriole at several places along the arteriolar length. The algorithm then “filled in” these marks to generate two continuous contours that “hugged” these edges. A computer routine used these contours to determine luminal diameters every 20 μm. Based on these diameters and on Poiseuille's law, the routine also estimated the hemodynamic resistance of the blood vessel. To demonstrate the usefulness of the method, we examined the character of spatial decay of KCl-induced conducted constriction along ∼500-μm-long arteriolar segments and the KCl-induced increase in hemodynamic resistance computed for these segments. The decay was only modestly fitted by a simple exponential, and the computed increase in resistance (i.e., 5- to 70-fold) was only modestly predicted by resistance increase based on our mathematical model involving measurements at two arteriolar sites (Tyml K, Wang X, Lidington D, and Oullette Y. Am J Physiol Heart Circ Physiol 281: H1397–H1406, 2001). We conclude that our method provides quick, reliable serial diameter measurements. Because the change in hemodynamic resistance could serve as a sensitive index of conducted response, use of this index in studies of conducted response may lead to new mechanistic insights on the response.

Lipopolysaccharide reduces intercellular coupling in vitro and arteriolar conducted response in vivo

Our recent in vitro study (Lidington et al. J Cell Physiol 185: 117–125, 2000) suggested that lipopolysaccharide (LPS) reduces communication along blood vessels. The present investigation extended this study to determine whether any effect of LPS and/or inflammatory cytokines [tumor necrosis factor-α, interleukin (IL)-1β, and IL-6] on endothelial cell coupling in vitro could also be demonstrated for an arteriolar conducted response in vivo. Using an electrophysiological approach in monolayers of microvascular endothelial cells, we found that LPS (10 μg/ml) but not these cytokines reduced intercellular conductance ( c i) (an index of cell communication) and that LPS together with these cytokines did not further reduce c i. Also, c i was restored after LPS washout, and the LPS-induced reduction was prevented by protein tyrosine kinase (PTK) inhibitors (1.5 μM Tyr A9 and 10 nM PP-2). In our in vivo experiments in arterioles of the mouse cremaster muscle, local electrical stimulation evoked vasoconstriction that conducted along arterioles. LPS in the muscle superfusate did not alter local vasoconstriction but reduced the conducted response. Washout of LPS restored the conducted response, whereas PTK inhibitors prevented the effect of LPS. On the basis of a newly developed mathematical model, the LPS-induced reduction in conducted response was predicted to reduce the arteriolar ability to increase resistance to blood flow. We conclude that LPS can reduce communication in in vitro and in vivo systems comparably in a reversible and tyrosine kinase-dependent manner. Based on literature and present results, we suggest that LPS may compromise microvascular hemodynamics at both the arteriolar responsiveness and the conduction levels.

Cumulative conducted vasodilation within a single arteriole and the maximum conducted response

The vascular network functions to distribute blood flow to the tissues that require it, and conducted vasodilation may facilitate this function. Experiments on arterioles in anesthetized hamster cheek pouch modeled the conducted responses that may come from a series of neighboring capillary modules and determined whether cumulative conducted responses could thereby maximally dilate upstream arterioles. Methacholine (10(-5) M) was simultaneously microapplied on an arteriole (resting diameter, approximately 22 microns; maximum diameter, approximately 47 microns) from one to four micropipettes spaced 100 microns apart, and with each added pipette the conducted dilation increased (up to a maximum dilation of approximately 5 microns). Increasing the methacholine 10-fold (10(-4) M) did not further increase the conducted response. The conducted response could also not be increased by lengthening the duration of microapplication. Yet, dilations that were not cumulative along a single arteriole became cumulative when initiated instead on adjacent arterioles. Therefore, these data demonstrate that conducted dilation along a single arteriole is limited and, if this model is correct, suggest that neighboring capillary modules may communicate only a limited conducted response to the network.

Components of methacholine-initiated conducted vasodilation are unaffected by arteriolar pressure

Conducted vasodilation occurs remotely from a site of microapplication of a drug. Intravascular pressure is required for a conducted response in vivo, yet in vitro studies in unpressurized arterioles show pressure is not essential. To determine how pressure affects conducted vasodilation, intra-arteriolar pressure was controlled within an in situ isolated segment (average length 950 +/- 96 microns, average baseline diameter 28 +/- 2.1 microns) of arterioles in the hamster cheek pouch. Methacholine (10(-4) M, 5 s) was microapplied either onto the isolated segment or remotely, with local and conducted vasodilation measured at both locations. Increasing pressure in the lumen of the segment (0-80 cmH2O) increased the segment local dilation to methacholine, and the segment-conducted dilation plateaued (at 4.1 +/- 0.8 micron) when segment pressure reached 20 cmH2O. Any local (16 +/- 1.5 microns) and conducted (4.4 +/- 1.3 microns) dilations viewed outside the segment were unaffected by segment pressure and persisted in its absence. Thus segment pressure affected only electromechanical transduction of the conducted response. Thus vasomotor signals move throughout the vasculature regardless of tone, but tone is essential to transduce the response.

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)