The role of pannexin/purinergic signaling in intervascular communication from capillaries during skeletal muscle contraction in male Golden hamsters.

We sought to determine the physiological relevance of pannexin/purinergic-dependent signaling in mediating conducted vasodilation elicited by capillary stimulation through skeletal muscle contraction. Using hamster cremaster muscle and intravital microscopy we stimulated capillaries through local muscle contraction while observing the associated upstream arteriole. Capillaries were stimulated with muscle contraction at low and high contraction (6 and 60CPM) and stimulus frequencies (4 and 40 Hz) in the absence and presence of pannexin blocker mefloquine (MEF; 10-5 M), purinergic receptor antagonist suramin (SUR 10-5 M) and gap-junction uncoupler halothane (HALO, 0.07%) applied between the capillary stimulation site and the upstream arteriolar observation site. Conducted vasodilations elicited at 6CPM were inhibited by HALO while vasodilations at 60CPM were inhibited by MEF and SUR. The conducted response elicited at 4 Hz was inhibited by MEF while the vasodilation at 40 Hz was unaffected by any blocker. Therefore, upstream vasodilations resulting from capillary stimulation via muscle contraction are dependent upon a pannexin/purinergic-dependent pathway that appears to be stimulation parameter-dependent. Our data highlight a physiological importance of the pannexin/purinergic pathway in facilitating communication between capillaries and upstream arteriolar microvasculature and, consequently, indicating that this pathway may play a crucial role in regulating blood flow in response to skeletal muscle contraction.

Mutant Cx30-A88V mice exhibit hydrocephaly and sex-dependent behavioral abnormalities, implicating a functional role for Cx30 in the brain.

Connexin 30 (Cx30; also known as Gjb6 when referring to the mouse gene) is expressed in ependymal cells of the brain ventricles, in leptomeningeal cells and in astrocytes rich in connexin 43 (Cx43), leading us to question whether patients harboring GJB6 mutations exhibit any brain anomalies. Here, we used mice harboring the human disease-associated A88V Cx30 mutation to address this gap in knowledge. Brain Cx30 levels were lower in male and female Cx30A88V/A88V mice compared with Cx30A88V/+ and Cx30+/+ mice, whereas Cx43 levels were lower only in female Cx30 mutant mice. Characterization of brain morphology revealed a disrupted ependymal cell layer, significant hydrocephalus and enlarged ventricles in 3- to 6-month-old adult male and female Cx30A88V/A88V mice compared with Cx30A88V/+ or Cx30+/+ sex-matched littermate mice. To determine the functional significance of these molecular and morphological changes, we investigated a number of behavioral activities in these mice. Interestingly, only female Cx30A88V/A88V mice exhibited abnormal behavior compared with all other groups. Cx30A88V/A88V female mice demonstrated increased locomotor and exploratory activity in both the open field and the elevated plus maze. They also exhibited dramatically reduced ability to learn the location of the escape platform during Morris water maze training, although they were able to swim as well as other genotypes. Our findings suggest that the homozygous A88V mutation in Cx30 causes major morphological changes in the brain of aging mice, possibly attributable to an abnormal ependymal cell layer. Remarkably, these changes had a more pronounced consequence for cognitive function in female mice, which is likely to be linked to the dysregulation of both Cx30 and Cx43 levels in the brain.

Capillaries communicate with the arteriolar microvascular network by a pannexin/purinergic-dependent pathway in hamster skeletal muscle.

We sought to determine if a pannexin/purinergic-dependent intravascular communication pathway exists in skeletal muscle microvasculature that facilitates capillary communication with upstream arterioles that control their perfusion. Using the hamster cremaster muscle and intravital microscopy, we locally stimulated capillaries and observed the vasodilatory response in the associated upstream 4A arteriole. We stimulated capillaries with vasodilators relevant to muscle contraction: 10-6 M S-nitroso-N-acetyl-dl-penicillamine (SNAP; nitric oxide donor), 10-6 M adenosine, 10 mM potassium chloride, 10-5 M pinacidil, as well as a known initiator of gap-junction-dependent intravascular communication, acetylcholine (10-5 M), in the absence and the presence of the purinergic membrane receptor blocker suramin (10-5 M), pannexin blocker mefloquine (2 × 10-5 M), or probenecid (5 × 10-6 M) and gap-junction inhibitor halothane (0.07%) applied in the transmission pathway, between the capillary stimulation site and the upstream 4A observation site. Potassium chloride, SNAP, and adenosine-induced upstream vasodilations were significantly inhibited by suramin, mefloquine, and probenecid but not halothane, indicating the involvement of a pannexin/purinergic-dependent signaling pathway. Conversely, SNAP-induced upstream vasodilation was only inhibited by halothane indicating that communication was facilitated by gap junctions. Both pinacidil and acetylcholine were inhibited by suramin but only acetylcholine was inhibited by halothane. These data demonstrate the presence of a pannexin/purinergic-dependent communication pathway between capillaries and upstream arterioles controlling their perfusion. This pathway adds to the gap-junction-dependent pathway that exists at this vascular level as well. Given that vasodilators relevant to muscle contraction can use both of these pathways, our data implicate the involvement of both pathways in the coordination of skeletal muscle blood flow.NEW & NOTEWORTHY Blood flow control during increased metabolic demand in skeletal muscle is not fully understood. Capillaries have been implicated in controlling blood flow to active skeletal muscle, but how capillaries communicate to the arteriolar vascular network is not clear. Our study uncovers a novel pathway through which capillaries can communicate to upstream arterioles to cause vasodilation and therefore control perfusion. This work implicates a new vascular communication pathway in blood flow control in skeletal muscle.

Ablation of both Cx40 and Panx1 results in similar cardiovascular phenotypes exhibited in Cx40 knockout mice.

Connexins (Cxs) and pannexins (Panxs) are highly regulated large-pore channel-forming proteins that participate in cellular communication via small molecular exchange with the extracellular microenvironment, or in the case of connexins, directly between cells. Given the putative functional overlap between single membrane-spanning connexin hemichannels and Panx channels, and cardiovascular system prevalence, we generated the first Cx40-/-Panx1-/- mouse with the anticipation that this genetic modification would lead to a severe cardiovascular phenotype. Mice null for both Cx40 and Panx1 produced litter sizes and adult growth progression similar to wild-type (WT), Cx40-/- and Panx1-/- mice. Akin to Cx40-/- mice, Cx40-/-Panx1-/- mice exhibited cardiac hypertrophy and elevated systolic, diastolic, and mean arterial blood pressure compared with WT and Panx1-/- mice; however assessment of left ventricular ejection fraction and fractional shortening revealed no evidence of cardiac dysfunction between groups. Furthermore, Cx40-/-, Panx1-/-, and Cx40-/-Panx1-/- mice demonstrated impaired endothelial-mediated vasodilation of aortic segments to increasing concentrations of methacholine (MCh) compared with WT, highlighting roles for both Cx40 and Panx1 in vascular endothelial cell (EC) function. Surprisingly, elevated kidney renin mRNA expression, plasma renin activity, and extraglomerular renin-producing cell populations found in Cx40-/- mice was further exaggerated in double knockout mice. Thus, while gestation and gross development were conserved in Cx40-/-Panx1-/- mice, they exhibit cardiac hypertrophy, hypertension, and impaired endothelial-mediated vasodilation that phenocopies Cx40-/- mice. Nevertheless, the augmented renin homeostasis observed in the double knockout mice suggests that both Cx40 and Panx1 may play an integrative role.

Neuropeptide Y1 and alpha-1 adrenergic receptor-mediated decreases in functional vasodilation in gluteus maximus microvascular networks of prediabetic mice.

Prediabetes is associated with impaired contraction-evoked dilation of skeletal muscle arterioles, which may be due to increased sympathetic activity accompanying this early stage of diabetes disease. Herein, we sought to determine whether blunted contraction-evoked vasodilation resulted from enhanced sympathetic neuropeptide Y1 receptor (Y1R) and alpha-1 adrenergic receptor (α1R) activation. Using intravital video microscopy, second-, third-, and fourth-order (2A, 3A, and 4A) arteriolar diameters were measured before and following electrical field stimulation of the gluteus maximus muscle (GM) in prediabetic (PD, Pound Mouse) and control (CTRL, c57bl6, CTRL) mice. Baseline diameter was similar between groups; however, single tetanic contraction (100 Hz; 400 and 800 msec) and sustained rhythmic contraction (2 and 8 Hz, 30 sec) evoked rapid onset vasodilation and steady-state vasodilatory responses that were blunted by 50% or greater in PD versus CTRL. Following Y1R and α1R blockade with sympathetic antagonists BIBP3226 and prazosin, contraction-evoked arteriolar dilation in PD was restored to levels observed in CTRL. Furthermore, arteriolar vasoconstrictor responses to NPY (10-13 -10-8  mol/L) and PE (10-9 -10-5  mol/L) were greater in PD versus CTRL at higher concentrations, especially at 3A and 4A. These findings suggest that contraction-evoked vasodilation in PD is blunted by Y1R and α1R receptor activation throughout skeletal muscle arteriolar networks.