Continuous cerebral blood flow is essential for neuronal survival, but whether vascular tone influences resting neuronal function is not known. Using a multidisciplinary approach in both rat and mice brain slices, we determined whether flow/pressure-evoked increases or decreases in parenchymal arteriole vascular tone, which result in arteriole constriction and dilation, respectively, altered resting cortical pyramidal neuron activity. We present evidence for intercellular communication in the brain involving a flow of information from vessel to astrocyte to neuron, a direction opposite to that of classic neurovascular coupling and referred to here as vasculo-neuronal coupling (VNC). Flow/pressure increases within parenchymal arterioles increased vascular tone and simultaneously decreased resting pyramidal neuron firing activity. On the other hand, flow/pressure decreases evoke parenchymal arteriole dilation and increased resting pyramidal neuron firing activity. InGLAST-CreERT2;R26-lsl-GCaMP3mice, we demonstrate that increased parenchymal arteriole tone significantly increased intracellular calcium in perivascular astrocyte processes, the onset of astrocyte calcium changes preceded the inhibition of cortical pyramidal neuronal firing activity. During increases in parenchymal arteriole tone, the pyramidal neuron response was unaffected by blockers of nitric oxide, GABA_A, glutamate, or ecto-ATPase. However, VNC was abrogated by TRPV4 channel, GABA_B, as well as an adenosine A₁receptor blocker. Differently to pyramidal neuron responses, increases in flow/pressure within parenchymal arterioles increased the firing activity of a subtype of interneuron. Together, these data suggest that VNC is a complex constitutive active process that enables neurons to efficiently adjust their resting activity according to brain perfusion levels, thus safeguarding cellular homeostasis by preventing mismatches between energy supply and demand.SIGNIFICANCE STATEMENTWe present evidence for vessel-to-neuron communication in the brain slice defined here as vasculo-neuronal coupling. We showed that, in response to increases in parenchymal arteriole tone, astrocyte intracellular Ca²⁺increased and cortical neuronal activity decreased. On the other hand, decreasing parenchymal arteriole tone increased resting cortical pyramidal neuron activity. Vasculo-neuronal coupling was partly mediated by TRPV4 channels as genetic ablation, or pharmacological blockade impaired increased flow/pressure-evoked neuronal inhibition. Increased flow/pressure-evoked neuronal inhibition was blocked in the presence of adenosine A1 receptor and GABA_Breceptor blockade. Results provide evidence for the concept of vasculo-neuronal coupling and highlight the importance of understanding the interplay between basal CBF and resting neuronal activity.

Enhanced vascular arginase activity impairs endothelium-dependent vasorelaxation by decreasing l-arginine availability to endothelial nitric oxide (NO) synthase, thereby reducing NO production. Elevated angiotensin II (ANG II) is a key component of endothelial dysfunction in many cardiovascular diseases and has been linked to elevated arginase activity. We determined signaling mechanisms by which ANG II increases endothelial arginase function. Results show that ANG II (0.1 μM, 24 h) elevates arginase activity and arginase I expression in bovine aortic endothelial cells (BAECs) and decreases NO production. These effects are prevented by the arginase inhibitor BEC (100 μM). Blockade of ANG II AT₁receptors or transfection with small interfering RNA (siRNA) for Gα12 and Gα13 also prevents ANG II-induced elevation of arginase activity, but siRNA for Gαq does not. ANG II also elevates active RhoA levels and induces phosphorylation of p38 MAPK. Inhibitors of RhoA activation (simvastatin, 0.1 μM) or Rho kinase (ROCK) (Y-27632, 10 μM; H1152, 0.5 μM) block both ANG II-induced elevation of arginase activity and phosphorylation of p38 MAPK. Furthermore, pretreatment of BAECs with p38 inhibitor SB-202190 (2 μM) or transfection with p38 MAPK siRNA prevents ANG II-induced increased arginase activity/expression and maintains NO production. Additionally, inhibitors of p38 MAPK (SB-203580, 5 μg·kg⁻¹·day⁻¹) or arginase (ABH, 8 mg·kg⁻¹·day⁻¹) or arginase gene knockout in mice prevents ANG II-induced vascular endothelial dysfunction and associated enhancement of arginase. These results indicate that ANG II increases endothelial arginase activity/expression through Gα12/13 G proteins coupled to AT₁receptors and subsequent activation of RhoA/ROCK/p38 MAPK pathways leading to endothelial dysfunction.

Reduced production of nitric oxide (NO) is one of the first indications of endothelial dysfunction and precedes overt cardiovascular disease. Increased expression of Arginase has been proposed as a mechanism to account for diminished NO production. Arginases consume l-arginine, the substrate for endothelial nitric oxide synthase (eNOS), and l-arginine depletion is thought to competitively reduce eNOS-derived NO. However, this simple relationship is complicated by the paradox that l-arginine concentrations in endothelial cells remain sufficiently high to support NO synthesis. One mechanism proposed to explain this is compartmentalization of intracellular l-arginine into distinct, poorly interchangeable pools. In the current study, we investigated this concept by targeting eNOS and Arginase to different intracellular locations within COS-7 cells and also BAEC. We found that supplemental l-arginine and l-citrulline dose-dependently increased NO production in a manner independent of the intracellular location of eNOS. Cytosolic arginase I and mitochondrial arginase II reduced eNOS activity equally regardless of where in the cell eNOS was expressed. Similarly, targeting arginase I to disparate regions of the cell did not differentially modify eNOS activity. Arginase-dependent suppression of eNOS activity was reversed by pharmacological inhibitors and absent in a catalytically inactive mutant. Arginase did not directly interact with eNOS, and the metabolic products of arginase or downstream enzymes did not contribute to eNOS inhibition. Cells expressing arginase had significantly lower levels of intracellular l-arginine and higher levels of ornithine. These results suggest that arginases inhibit eNOS activity by depletion of substrate and that the compartmentalization of l-arginine does not play a major role.

Arginase can cause vascular dysfunction by competing with nitric oxide synthase for l-arginine and by increasing cell proliferation and collagen formation, which promote vascular fibrosis/stiffening. We have shown that increased arginase expression/activity contribute to vascular endothelial cell (EC) dysfunction. Here, we examined the roles of the two arginase isoforms, arginase I and II (AI and AII, respectively), in this process. Experiments were performed using streptozotocin-induced diabetic mice: wild-type (WT) mice and knockout mice lacking the AII isoform alone (AI^+/+AII^−/−) or in combination with partial deletion of AI (AI^+/−AII ^−/−). EC-dependent vasorelaxation of aortic rings and arterial fibrosis and stiffness were assessed in relation to arginase activity and expression. Diabetes reduced mean EC-dependent vasorelaxation markedly in diabetic WT and AI^+/+AII^−/− aortas (53% and 44% vs. controls, respectively) compared with a 27% decrease in AI^+/−AII ^−/− vessels. Coronary fibrosis was also increased in diabetic WT and AI^+/+AII^−/− mice (1.9- and 1.7-fold vs. controls, respectively) but was not altered in AI^+/−AII ^−/− diabetic mice. Carotid stiffness was increased by 142% in WT diabetic mice compared with 51% in AI^+/+AII^−/− mice and 19% in AI^+/−AII ^−/− mice. In diabetic WT and AI^+/+AII^−/− mice, aortic arginase activity and AI expression were significantly increased compared with control mice, but neither parameter was altered in AI^+/−AII ^−/− mice. In summary, AI^+/−AII ^−/− mice exhibit better EC-dependent vasodilation and less vascular stiffness and coronary fibrosis compared with diabetic WT and AI^+/+AII^−/− mice. These data indicate a major involvement of AI in diabetes-induced vascular dysfunction.

Functional hyperemia is the regional increase in cerebral blood flow upon increases in neuronal activity which ensures that the metabolic demands of the neurons are met. Hypertension is known to impair the hyperemic response; however, the neurovascular coupling mechanisms by which this cerebrovascular dysfunction occurs have yet to be fully elucidated. To determine whether altered cortical parenchymal arteriole function or astrocyte signaling contribute to blunted neurovascular coupling in hypertension, we measured parenchymal arteriole reactivity and vascular smooth muscle cell Ca²+ dynamics in cortical brain slices from normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. We found that vasoconstriction in response to the thromboxane A₂ receptor agonist U46619 and basal vascular smooth muscle cell Ca²⁺ oscillation frequency were significantly increased in parenchymal arterioles from SHR. In perfused and pressurized parenchymal arterioles, myogenic tone was significantly increased in SHR. Although K⁺-induced parenchymal arteriole dilations were similar in WKY and SHR, metabotropic glutamate receptor activation-induced parenchymal arteriole dilations were enhanced in SHR. Further, neuronal stimulation-evoked parenchymal arteriole dilations were similar in SHR and WKY. Our data indicate that neurovascular coupling is not impaired in SHR, at least at the level of the parenchymal arterioles.

Background: Transcutaneous spinal stimulation (TSS) is a noninvasive stimulation approach to modulate spinal reflex circuit excitability after spinal cord injury (SCI) Posterior root muscle (PRM) reflexes can be used to characterize the change in excitability of spinal reflex circuits after TSS; these responses are likely influenced by stimulus parameters. Methods: We compared PRM reflex responses to 3 TSS conditions: single-site continuous (SS-CONT), single-site burst (SS-BURST), and dual-site continuous (DS-CONT). Stimulation (frequency: 50 Hz, intensity: 80% soleus reflex threshold[RT]) was delivered for 30 min. The cathode was placed over the thoracic spine (T11–T12) and anodes placed paraumbilically; a second cathode over the lumbar spine (L1/2 or L2/3) was used for DS-CONT. PRM reflex responses in the soleus were elicited by paired 1 ms monophasic conditioning–test stimuli at a 50 ms interstimulus interval via the T11–12 cathode and paraumbilical anodes. Soleus PRM reflex indices included RT, response amplitude at 1.2xRT (RA_1.2xRT), slope, area under the input–output curve (AUC). Paired-pulse indices were collected, including paired-pulse depression (PPD) and depression of the area under the curve (AUC_dep). To assess the correlation between biomechanical and electrophysiologic measures of soleus spasticity, the ankle clonus drop test first drop excursion (FDE) was measured. All indices were measured at baseline and immediately post-intervention. Results: In whole-group analyses, PPD and AUC_dep were significantly decreased. Significant decreases in PPD and AUC_dep were identified only after the SS-CONT condition. No significant changes were identified in other PRM reflex indices after any of the 3 TSS conditions. No relationships between baseline FDE and any PRM reflex parameter were identified at baseline. Conclusions: With stimulation intensity of 80% soleus RT, modulation of targeted spinal reflex circuits was observed only in the SS-CONT condition when the response of the conditioning and test stimuli were considered. In addition, stretch-induced spasticity of the soleus may not be consistent with electrophysiologic testing.