Evidence for Capillary Arteriolar Communication

In Vivo Work

The majority of experimental evidence for communication along the capillary is indirect. Using amphibian and mammalian skeletal muscle preparations, our laboratory has shown that micropipette application of a minute amount of various vasoactive agents (norepinephrine, phenylephrine, acetylcholine, bradykinin, adenosine analog NECA, KCl) on capillaries 300 to 500 mm away from the feeding arteriole causes constriction/dilation of this arteriole and ensuing reduction/increase in blood flow in capillaries fed by this arteriole (Figure 1). Simple diffusion failed to explain these responses since interventions that do not alter the diffusional process [e.g., treatment of the capillary midpoint with GJ uncoupler, injury at this midpoint (Figure 1), or systemic application of GJ uncoupler] inhibited these responses. These experiments suggested that the capillary itself could sense vasoactive agents and that it could provide a commu-

Figure 1 Schematic diagram of experimental arrangement for distal stimulation of capillaries. In this diagram, capillaries are oriented horizontally, run in parallel to each other, and are fed by arteriole "A." Arrows indicate direction of blood flow while dots represent physical junction between microvessels. During the experiment, a minute amount of vasoactive agent is placed on capillary by means of a glass micropipette, 300 to 500 mm away from the arteriole. Responses are measured in terms of changes in arteriolar diameter or changes in red blood cell flow in the stimulated capillary (or any capillary supplied by the terminal end of the arteriole). Local damage of the stimulated capillary at midpoint (i.e., indicated by dotted circle), or pretreatment of the midpoint by gap junction uncoupler, inhibits the arteriolar/blood flow response to the distal capillary stimulus [3, 4]. (Adapted from Ref. [3].)

nication feedback path between the site of locally produced/applied agents and the arteriole. Further, stimulation of two capillaries fed by the same arteriole yielded addi-tive/subtractive responses at the arteriolar level, suggesting that the capillary bed could function as a dispersed sensor within the tissue that is able to integrate metabolic signals within this tissue into an "overall" response at the arteriolar level [5].

Although the capillary sensing/communication phenomenon characterized by the above observations gave rise to a new concept of local blood flow control, the physiological significance of this phenomenon is not clear. To this end, two laboratories provided data relevant to this issue. First, Berg and coworkers [6] reported that, in hamster cremaster muscle, locally induced contraction of muscle bundle unit supplied by capillary bed via a single terminal arteriole caused conducted dilation along this arteriole. A local application of a GJ uncoupler on this arteriole prevented this dilation. Since mechanical tugging of the muscle unit did not result in conducted dilation, it was speculated that the capillary bed was involved in the transmission of contraction-induced vasoactive metabolite(s) to the arteriole. Thus, capillary-arteriolar communication could be involved in matching of the dilation-mediated increase in blood flow to the contraction-induced metabolic demand. Second, Collins and coworkers [7] reported that intravenular application of ATP (1 mM) via micropipette caused an upstream arteriolar dilation. ATP at this concentration has been reported to be released from red blood cells in response to low pH and hypoxia in venous blood. In order to explain the upstream dilation, these workers proposed that ATP stimulated endothelial P2Y and P2U receptors in venules to initiate conducted vascular response across the capillary bed to the arteriole. Thus, capillary endothelium could participate in communication of oxygen sensing signals between the venular and arteriolar ends of the microvasculature.

Unlike in capillaries, conduction of local agonist-induced responses is well characterized for the arteriole. Using recording electrodes inserted into the arteriolar wall at the local site of agonist application and at an upstream site, agonist-induced hyper/depolarizations are seen to spread electrotonically along the arteriolar endothelium. Since the capillary endothelium forms a continuous layer with the arteriolar endothelium, the mechanism of capillary sensing/communication has been assumed to be of a similar nature, that is, involving an electrotonic spread of locally induced hyper/depolarizations. Because of technical difficulties, however, direct electrical recordings from the capillary endothelium have not yet been carried out to confirm this mechanism. To date, an indirect approach has been used instead. Using a voltage-sensitive dye loaded into the capillary endothelium, hyperpolarization and depolarization of endothelial cells were observed after local application of agonists [8]. Employing this method, local depolarization initiated at the upstream arteriole site conducted to a downstream capillary site, demonstrating that electrotonic communication can occur along the capillary endothelium [9].

Figure 1 Schematic diagram of experimental arrangement for distal stimulation of capillaries. In this diagram, capillaries are oriented horizontally, run in parallel to each other, and are fed by arteriole "A." Arrows indicate direction of blood flow while dots represent physical junction between microvessels. During the experiment, a minute amount of vasoactive agent is placed on capillary by means of a glass micropipette, 300 to 500 mm away from the arteriole. Responses are measured in terms of changes in arteriolar diameter or changes in red blood cell flow in the stimulated capillary (or any capillary supplied by the terminal end of the arteriole). Local damage of the stimulated capillary at midpoint (i.e., indicated by dotted circle), or pretreatment of the midpoint by gap junction uncoupler, inhibits the arteriolar/blood flow response to the distal capillary stimulus [3, 4]. (Adapted from Ref. [3].)

Another group has used a calcium-sensitive dye in venular capillaries of perfused lung. Spontaneous calcium waves were seen to spread along the capillary wall [10]. That this spread was inhibited by a GJ uncoupler indicated that capillary endothelial cells could communicate via gap junctions in vivo.

In Vitro Work

There is considerable in vitro evidence that microvascu-lar endothelial cells (i.e., the major component of the capillary wall) can communicate electrically via gap junctions. When a single endothelial cell grown in a culture dish is connected to a patch-clamp electrode, its measured input electrical conductance is much smaller than that obtained for a confluent cell monolayer (i.e., indicating substantial intercellular coupling within the monolayer). Alternatively, when a cell in a monolayer is injected with brief pulses of electrical current, rapid electrotonic spread of these pulses through the monolayer is seen (i.e., pulse amplitude decreases with distance along the monolayer). Cultured microvascular endothelial cells have been shown to hyper/depolarize in response to application of vasoactive agents. (Note: with increased cell passaging, the responsiveness to many agents disappears, most likely due to the loss of expression of the appropriate receptors.) We have shown that this hyper/depolarization also communicated along the monolayer, but with a substantial spatial decay (only 20 to 30% of the local membrane potential change was seen at 300 mm distance) [11].

Recently, our laboratory has employed an alternative in vitro model to study endothelial cell-to-cell communication. Instead of monolayers, microvascular endothelial cells (origin: rat skeletal muscle) were grown in Matrigel matrix as "capillary-like" structures (i.e., 300 to 500 mm long with about four cells per 100 mm). A local, agonist-induced hyper/depolarization communicated electrotonically along these structures with a much smaller decay (i.e., 70% of the local response at 300 mm) than in monolayers. Thus, communication along these structures better mimicked the one-dimensional communication along in vivo microvessels than the two-dimensional communication seen in monolayers [11]. In both models, GJ uncouplers inhibited communication. Since, in general, all three vascular connexins have been found in cultured endothelial cells, the available functional and structural evidence indicates that endothelial cells in vitro have the fundamental ability to respond to a variety of vasoactive stimuli and to communicate this response (e.g., hyper/depolarization) rapidly along the microvascular endothelium, including the capillary endothelium.

Essentials of Human Physiology

Essentials of Human Physiology

This ebook provides an introductory explanation of the workings of the human body, with an effort to draw connections between the body systems and explain their interdependencies. A framework for the book is homeostasis and how the body maintains balance within each system. This is intended as a first introduction to physiology for a college-level course.

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