Abstract-During exercise, local mechanisms in tissues cause arterioles to rapidly dilate to increase blood flow to tissues to meet the metabolic demands of contracting muscle. Despite decades of study, the mechanisms underlying this important aspect of blood flow control are still far from clear. We now report a novel mechanism wherein fibronectin fibrils in connective tissue matrices transduce signals from contracting skeletal muscle to local blood vessels to increase blood flow. Using intravital microscopy, we show that local vasodilation in response to skeletal muscle contraction is specifically inhibited by an antibody that recognizes the matricryptic site in the first type III repeat of fibronectin (FNIII-1). In the absence of skeletal muscle contraction, direct application of FNIII-1-containing fibronectin fragments to cremaster muscle arterioles in situ, triggered a rapid, specific, and reversible local dilation that was mediated by nitric oxide and required the cryptic, heparin-binding sequence of FNIII-1. Furthermore, application of function-blocking FNIII-1 peptides to cremaster muscle arterioles rapidly and specifically decreased their diameter, indicating that the matricryptic site of fibronectin also contributes to resting vascular tone. Alexa fluor 488 -labeled fibronectin, administered intravenously, was rapidly assembled into elongated, branching fibrils in the extracellular matrix of intact cremaster muscle, demonstrating active polymerization of fibronectin in areas adjacent to blood vessels. Together, these data provide the first evidence that a matricryptic, heparin-binding site within fibronectin fibrils of adult connective tissue plays a dynamic role in regulating both vascular responses and vascular tone.

Key pointsr The adenosine-dependent component of functional dilation of small resistance arterioles acts via A 2A receptors located on endothelium to activate K ATP channels on associated vascular smooth muscle.r A 2A receptors are G s -coupled, hence receptor occupancy should activate cAMP/protein kinase A (PKA) cell signalling pathways However, pre-exposure to adenosine alters the PKA dependence of the response and renders the vessel insensitive to PKA inhibition.r The adenosine pre-exposure effect is mimicked by pre-activation of PKA and is specific to adenosine, as the PKA dependence of dilation to isoproterenol (another G s -coupled agonist) is not affected by pre-exposure.r Activation of PKA alone does not induce dilation. An additional signalling mechanism, dependent on increased EC Ca 2+ via activation of cyclic nucleotide gated channels, is required together with PKA activation to produce A 2A -dependent vasodilation.r This novel identification of variability in signalling downstream from a single receptor to produce the same response may reflect a mechanism for integration of key homeostatic responses.Abstract Adenosine (ADO) is an endogenous vasodilatory purine widely recognized to be a significant contributor to functional hyperaemia. Despite this, many aspects of the mechanisms by which ADO induces dilation in small resistance arterioles are not established, or appear contradictory. These include: identification of the primary receptor subtype; its location on endothelial (EC) or vascular smooth muscle cells; whether ADO acts on K ATP channels in these resistance vessels; and the contribution of cAMP/protein kinase A (PKA) signalling to the response. In intravital microscopy studies of intact or EC-denuded skeletal muscle arterioles, we show that ADO acts via A 2A receptors located on ECs to produce vasodilation via activation of K ATP channels located on vascular smooth muscle cells. Importantly, we found that the signalling pathway involves cAMP as expected, but that a requirement for PKA activation is demonstrable only if the vessel is not pre-exposed to ADO. That is, PKA-dependent signalling varies with pre-exposure to ADO. Further, we show that PKA activation alone is not sufficient to dilate these arterioles; an additional EC calcium-dependent signalling mechanism is required for vasodilation to ADO. The ability of arterioles in situ to respond to occupancy of a specific receptor by utilizing different cell signalling pathways under different conditions to produce the same response allows the arteriole to respond to key homeostatic requirements using more than a single signalling mechanism. Clearly, this is likely to be physiologically advantageous, but the role for this signalling flexibility in the integrated arteriolar response that underlies functional hyperaemia will require further exploration.

Key pointsr The local arteriolar dilatation produced by contraction of skeletal muscle is dependent upon multiple signalling mechanisms.r In addition to the many metabolic signals that mediate this vasodilatation, we show here that the extracellular matrix protein fibronectin also contributes to the response. Abstract Recent studies in contracting skeletal muscle have shown that functional vasodilatation in resistance arterioles has an endothelial cell (EC)-dependent component, and, separately have shown that the extracellular matrix protein fibronectin (FN) contributes to functional dilatation in these arterioles. Here we test the hypotheses that (i) the matricryptic heparin-binding region of the first type III repeat of fibrillar FN (FNIII1H) mediates vasodilatation, and (ii) this response is EC dependent. Engineered FN fragments with differing (defined) heparin-and integrin-binding capacities were applied directly to resistance arterioles in cremaster muscles of anaesthetized (pentobarbital sodium, 65 mg kg −1 ) mice. Both FNIII1H,8-10 and FNIII1H induced dilatations (12.2 ± 1.7 μm, n = 12 and 17.2 ± 2.4 μm, n = 14, respectively) whereas mutation of the active sequence (R 613 WRPK) of the heparin binding region significantly diminished the dilatation (3.2 ± 1.8 μm, n = 10). Contraction of skeletal muscle fibres via electrical field stimulation produced a vasodilatation (19.4 ± 1.2 μm, n = 12) that was significantly decreased (to 7.0 ± 2.7 μm, n = 7, P < 0.05) in the presence of FNIII1Peptide 6, which blocks extracellular matrix (ECM) FN and FNIII1H signalling. Furthermore, FNIII1H,[8][9][10] and FNIII1H applied to EC-denuded arterioles failed to produce any dilatation indicating that endothelium was required for the response. Finally, FNIII1H significantly increased EC Ca 2+ (relative fluorescence 0.98 ± 0.02 in controls versus 1.12 ± 0.05, n = 17, P < 0.05). Thus, we conclude that ECM FN-dependent vasodilatation is mediated by the heparin-binding (RWRPK) sequence of FNIII1 in an EC-dependent manner. Importantly, blocking this signalling sequence decreased the dilatation to skeletal muscle contraction, indicating that there is a physiological role for this FN-dependent mechanism.