Epidermal Growth Factor Receptors in Vascular Endothelial Cells Contribute to Functional Hyperemia in the Brain.

Functional hyperemia-activity-dependent increases in local blood perfusion-underlies the on-demand delivery of blood to regions of enhanced neuronal activity, a process that is crucial for brain health. Importantly, functional hyperemia deficits have been linked to multiple dementia risk factors, including aging, chronic hypertension, and cerebral small vessel disease (cSVD). We previously reported crippled functional hyperemia in a mouse model of genetic cSVD that was likely caused by depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) in capillary endothelial cells (EC) downstream of impaired epidermal growth factor receptor (EGFR) signaling. Here, using EC-specific EGFR-knockout (KO) mice, we directly examined the role of endothelial EGFR signaling in functional hyperemia, assessed by measuring increases in cerebral blood flow in response to contralateral whisker stimulation using laser Doppler flowmetry. Molecular characterizations showed that EGFR expression was dramatically decreased in freshly isolated capillaries from EC-EGFR-KO mice, as expected. Notably, whisker stimulation-induced functional hyperemia was significantly impaired in these mice, an effect that was rescued by administration of PIP2, but not by the EGFR ligand, HB-EGF. These data suggest that the deletion of the EGFR specifically in ECs attenuates functional hyperemia, likely via depleting PIP2 and subsequently incapacitating Kir2.1 channel functionality in capillary ECs. Thus, our study underscores the role of endothelial EGFR signaling in functional hyperemia of the brain.

Epidermal growth factor receptors in vascular endothelial cells contribute to functional hyperemia in the brain.

Functional hyperemia - activity-dependent increases in local blood perfusion - underlies the on-demand delivery of blood to regions of enhanced neuronal activity, a process that is crucial for brain health. Importantly, functional hyperemia deficits have been linked to multiple dementia risk factors, including aging, chronic hypertension, and cerebral small vessel disease (cSVD). We previously reported crippled functional hyperemia in a mouse model of genetic cSVD that was likely caused by depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) in capillary endothelial cells (EC) downstream of impaired epidermal growth factor receptor (EGFR) signaling. Here, using EC-specific EGFR-knockout (KO) mice, we directly examined the role of endothelial EGFR signaling in functional hyperemia, assessed by measuring increases in cerebral blood flow in response to contralateral whisker stimulation using laser Doppler flowmetry. Molecular characterizations showed that EGFR expression was dramatically decreased in freshly isolated capillaries from EC-EGFR-KO mice, as expected. Notably, whisker stimulation-induced functional hyperemia was significantly impaired in these mice, an effect that was rescued by exogenous administration of PIP2, but not by the EGFR ligand, HB-EGF. These data suggest that the deletion of the EGFR specifically in ECs depletes PIP2 and attenuates functional hyperemia, underscoring the central role of the endothelial EGFR signaling in cerebral blood flow regulation.

Differential restoration of functional hyperemia by antihypertensive drug classes in hypertension-related cerebral small vessel disease.

Dementia resulting from small vessel diseases (SVDs) of the brain is an emerging epidemic for which there is no treatment. Hypertension is the major risk factor for SVDs, but how hypertension damages the brain microcirculation is unclear. Here, we show that chronic hypertension in a mouse model progressively disrupts on-demand delivery of blood to metabolically active areas of the brain (functional hyperemia) through diminished activity of the capillary endothelial cell inward-rectifier potassium channel, Kir2.1. Despite similar efficacy in reducing blood pressure, amlodipine, a voltage-dependent calcium-channel blocker, prevented hypertension-related damage to functional hyperemia whereas losartan, an angiotensin II type 1 receptor blocker, did not. We attribute this drug class effect to losartan-induced aldosterone breakthrough, a phenomenon triggered by pharmacological interruption of the renin-angiotensin pathway leading to elevated plasma aldosterone levels. This hypothesis is supported by the finding that combining losartan with the aldosterone receptor antagonist eplerenone prevented the hypertension-related decline in functional hyperemia. Collectively, these data suggest Kir2.1 as a possible therapeutic target in vascular dementia and indicate that concurrent mineralocorticoid aldosterone receptor blockade may aid in protecting against late-life cognitive decline in hypertensive patients treated with angiotensin II type 1 receptor blockers.

Impaired Cerebral Autoregulation After Subarachnoid Hemorrhage: A Quantitative Assessment Using a Mouse Model.

Subarachnoid hemorrhage (SAH) is a common form of hemorrhagic stroke associated with high rates of mortality and severe disability. SAH patients often develop severe neurological deficits days after ictus, events attributed to a phenomenon referred to as delayed cerebral ischemia (DCI). Recent studies indicate that SAH-induced DCI results from a multitude of cerebral circulatory disturbances including cerebral autoregulation malfunction. Cerebral autoregulation incorporates the influence of blood pressure (BP) on arterial diameter in the homeostatic regulation of cerebral blood flow (CBF), which is necessary for maintaining constant brain perfusion during physiological swings in systemic BP. In this study, we quantitatively examined the impact of SAH on cerebral autoregulation using a mouse endovascular perforation model and a newly developed approach combining absolute and relative CBF measurements. This method enables a direct quantitative comparison of cerebral autoregulation between individual animals (e.g., SAH vs. control or sham-operated mice), which cannot be done solely using relative CBF changes by laser Doppler flowmetry. Here, absolute CBF was measured via injection of fluorescent microspheres at a baseline BP. In separate groups of animals, in vivo laser Doppler flowmetry was used to measure relative CBF changes over a range of BP using phlebotomy and the pressor phenylephrine to lower and raise BP, respectively. Absolute CBF measurements from microspheres were then used to calibrate laser Doppler measurements to calculate the relationship between CBF and BP, i.e., "cerebral autoregulation curves." Un-operated and sham-operated groups exhibited similar cerebral autoregulatory curves, showing comparable levels of relatively constant CBF over a range of BP from ~80 mmHg to ~130 mmHg. In contrast, SAH animals exhibited a narrower autoregulatory range of BP, which was primarily due to a decrease in the upper limit of BP whereby cerebral autoregulation was maintained. Importantly, SAH animals also exhibited a marked decrease in CBF throughout the entire range of BP. In sum, this study provides evidence of the dramatic reduction in cortical CBF and the diminished range of autoregulation after SAH. Furthermore, this novel methodology should pave the way for future studies examining pathological mechanisms and/or therapeutic strategies targeting impaired cerebral autoregulation, a pathology common to many cardiovascular and cerebrovascular disorders.

Endocrine Dysfunction Following Stroke.

Endocrine dysfunction is known to occur after traumatic brain injury. The purpose of this study was to examine the incidence of various endocrine dysfunctions after a stroke. The Taiwan National Health Insurance Research Database (NHIRD) was searched from 2001 to 2011 for patients with a diagnosis of stroke. Stroke patients were matched by diagnosis date, age, and sex to patients without a stroke. Cox proportional hazards regression analyses were performed to compare the incidence of goiter, acquired hypothyroidism, thyroiditis, pituitary dysfunction, and disorders of the adrenal glands between stroke and non-stroke patients. There were 131,951 patients in the stroke group, and 131,951 in the matched non- stroke group (mean age 66.1 ± 14.9 years). Stroke patients had significantly higher risk of acquired hypothyroidism (crude hazard ratio [cHR] = 1.65, 95% confidence interval [CI]: 1.44, 1.90; adjusted hazard ratio [aHR] = 1.65, 95% CI: 1.42, 1.91), pituitary dysfunction (cHR = 2.32, 95% CI: 1.79, 2.99; aHR = 1.92, 95% CI: 1.46, 2.52), and disorders of the adrenal glands (cHR = 1.79, 95% CI: 1.52, 2.12; aHR =1.62, 95% CI: 1.36, 1.92) than non-stroke patients. Pituitary dysfunction and disorders of the adrenal glands were found in both hemorrhagic stroke and ischemic stroke patients, while hypothyroidism was seen in ischemic stroke patients only. No significant association was found for goiter and thyroiditis. In conclusions, stroke survivors have an approximately 2-fold increased risk of developing acquired hypothyroidism, pituitary dysfunction, or disorders of the adrenal glands. These risks should be taken into account in the management of patients who have ischemic or hemorrhagic strokes. Graphical Abstract.

Tonic regulation of middle meningeal artery diameter by ATP-sensitive potassium channels.

Activation of ATP-sensitive potassium (KATP) channels in arterial smooth muscle (ASM) contributes to vasodilation evoked by a variety of endogenous and exogenous compounds. Although controversial, activation of KATP channels by neuropeptides such as calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase activating peptide (PACAP) in the trigeminovascular system, including the middle meningeal artery (MMA), has been linked to migraine headache. The objective of the current study was to determine if ongoing KATP channel activity also influences MMA diameter. In the absence of other exogenous compounds, the KATP channel inhibitors glibenclamide and PNU37883A induced constriction of isolated and pressurized MMAs. In contrast, KATP channel inhibition did not alter cerebral artery diameter. Consistent with tonic KATP activity in MMA, glibenclamide also induced ASM membrane potential depolarization and increased cytosolic Ca2+. Inhibitors of cAMP-dependent protein kinase (PKA) abolished basal KATP activation in MMA and caused a marked decrease in sensitivity to the synthetic KATP channel opener, cromakalim. In vivo MMA constriction in response to gibenclamide was observed using two-photon imaging of arterial diameter. Together these results indicate that PKA-mediated tonic KATP channel activity contributes to the regulation of MMA diameter.

The yin and yang of KV channels in cerebral small vessel pathologies.

Cerebral SVDs encompass a group of genetic and sporadic pathological processes leading to brain lesions, cognitive decline, and stroke. There is no specific treatment for SVDs, which progress silently for years before becoming clinically symptomatic. Here, we examine parallels in the functional defects of PAs in CADASIL, a monogenic form of SVD, and in response to SAH, a common type of hemorrhagic stroke that also targets the brain microvasculature. Both animal models exhibit dysregulation of the voltage-gated potassium channel, KV 1, in arteriolar myocytes, an impairment that compromises responses to vasoactive stimuli and impacts CBF autoregulation and local dilatory responses to neuronal activity (NVC). However, the extent to which this channelopathy-like defect ultimately contributes to these pathologies is unknown. Combining experimental data with computational modeling, we describe the role of KV 1 channels in the regulation of myocyte membrane potential at rest and during the modest increase in extracellular potassium associated with NVC. We conclude that PA resting membrane potential and myogenic tone depend strongly on KV 1.2/1.5 channel density, and that reciprocal changes in KV channel density in CADASIL and SAH produce opposite effects on extracellular potassium-mediated dilation during NVC.

Nifedipine Inhibition of High-Voltage Activated Calcium Channel Currents in Cerebral Artery Myocytes Is Influenced by Extracellular Divalent Cations.

Voltage-dependent calcium channels (VDCCs) play an essential role in regulating cerebral artery diameter and it is widely appreciated that the L-type VDCC, CaV1.2, encoded by the CACNA1C gene, is a principal Ca2+ entry pathway in vascular myocytes. However, electrophysiological studies using 10 mM extracellular barium ([Ba2+]o) as a charge carrier have shown that ~20% of VDCC currents in cerebral artery myocytes are insensitive to 1,4-dihydropyridine (1,4-DHP) L-type VDDC inhibitors such as nifedipine. Here, we investigated the hypothesis that the concentration of extracellular divalent cations can influence nifedipine inhibition of VDCC currents. Whole-cell VDCC membrane currents were obtained from freshly isolated rat cerebral artery myocytes in extracellular solutions containing Ba2+ and/or Ca2+. In the absence of [Ca2+]o, both nifedipine-sensitive and -insensitive calcium currents were observed in 10 mM [Ba2+]o. However, VDCC currents were abolished by nifedipine when using a combination of 10 mM [Ba2+]o and 100 µM [Ca2+]o. VDCC currents were also completely inhibited by nifedipine in either 2 mM [Ba2+]o or 2 mM [Ca2+]o. The biophysical characteristics of all recorded VDCC currents were consistent with properties of a high-voltage activated VDCC, such as CaV1.2. Further, VDCC currents recorded in 10 mM [Ba2+]o ± 100 µM [Ca2+]o or 2 mM [Ba2+]o exhibited similar sensitivity to the benzothiazepine L-type VDCC blocker, diltiazem, with complete current inhibition at 100 µM. These data suggest that nifedipine inhibition is influenced by both Ca2+ binding to an external site(s) on these channels and surface charge effects related to extracellular divalent cations. In sum, this work demonstrates that the extracellular environment can profoundly impact VDCC current measurements.

Inversion of neurovascular coupling after subarachnoid hemorrhage in vivo.

Subarachnoid hemorrhage (SAH) induces acute changes in the cerebral microcirculation. Recent findings ex vivo suggest neurovascular coupling (NVC), the process that increases cerebral blood flow upon neuronal activity, is also impaired after SAH. The aim of the current study was to investigate whether this occurs also in vivo. C57BL/6 mice were subjected to either sham surgery or SAH by filament perforation. Twenty-four hours later NVC was tested by forepaw stimulation and CO2 reactivity by inhalation of 10% CO2. Vessel diameter was assessed in vivo by two-photon microscopy. NVC was also investigated ex vivo using brain slices. Cerebral arterioles of sham-operated mice dilated to 130% of baseline upon CO2 inhalation or forepaw stimulation and cerebral blood flow (CBF) increased. Following SAH, however, CO2 reactivity was completely lost and the majority of cerebral arterioles showed paradoxical constriction in vivo and ex vivo resulting in a reduced CBF response. As previous results showed intact NVC 3 h after SAH, the current findings indicate that impairment of NVC after cerebral hemorrhage occurs secondarily and is progressive. Since neuronal activity-induced vasoconstriction (inverse NVC) is likely to further aggravate SAH-induced cerebral ischemia and subsequent brain damage, inverse NVC may represent a novel therapeutic target after SAH.

Acute changes in neurovascular reactivity after subarachnoid hemorrhage in vivo.

Subarachnoid hemorrhage causes acute and long-lasting constrictions of pial arterioles. Whether these vessels dilate normally to neuronal activity is of great interest since a mismatch between delivery and consumption of glucose and oxygen may cause additional neuronal damage. Therefore, we investigated neurovascular reactivity of pial and parenchymal arterioles after experimental subarachnoid hemorrhage. C57BL/6 mice were subjected to subarachnoid hemorrhage by filament perforation or sham surgery. Neurovascular reactivity was assessed 3 h later by forepaw stimulation or inhalation of 10% CO2 Diameters of cerebral arterioles were assessed using two-photon microscopy. Neurovascular coupling and astrocytic endfoot Ca2+ were measured in brain slices using two-photon and infrared-differential interference contrast microscopy. Vessels of sham-operated mice dilated normally to CO2 and forepaw stimulation. Three hours after subarachnoid hemorrhage, CO2 reactivity was completely lost in both pial and parenchymal arterioles, while neurovascular coupling was not affected. Brain slices studies also showed normal neurovascular coupling and a normal increase in astrocytic endfoot Ca2+ acutely after subarachnoid hemorrhage. These findings suggest that communication between neurons, astrocytes, and parenchymal arterioles is not affected in the first few hours after subarachnoid hemorrhage, while CO2 reactivity, which is dependent on NO signaling, is completely lost.

Traumatic Brain Injury Causes Endothelial Dysfunction in the Systemic Microcirculation through Arginase-1-Dependent Uncoupling of Endothelial Nitric Oxide Synthase.

Endothelial dysfunction is a hallmark of many chronic diseases, including diabetes and long-term hypertension. We show that acute traumatic brain injury (TBI) leads to endothelial dysfunction in rat mesenteric arteries. Endothelial-dependent dilation was greatly diminished 24 h after TBI because of impaired nitric oxide (NO) production. The activity of arginase, which competes with endothelial NO synthase (eNOS) for the common substrate l-arginine, were also significantly increased in arteries, suggesting that arginase-mediated depletion of l-arginine underlies diminished NO production. Consistent with this, substrate restoration by exogenous application of l-arginine or inhibition of arginase recovered endothelial function. Moreover, evidence for increased reactive oxygen species production, a consequence of l-arginine starvation-dependent eNOS uncoupling, was detected in endothelium and plasma. Collectively, our findings demonstrate endothelial dysfunction in a remote vascular bed after TBI, manifesting as impaired endothelial-dependent vasodilation, with increased arginase activity, decreased generation of NO, and increased O2- production. We conclude that blood vessels have a "molecular memory" of neurotrauma, 24 h after injury, because of functional changes in vascular endothelial cells; these effects are pertinent to understanding the systemic inflammatory response that occurs after TBI even in the absence of polytrauma.

Purinergic signaling triggers endfoot high-amplitude Ca2+ signals and causes inversion of neurovascular coupling after subarachnoid hemorrhage.

Neurovascular coupling supports brain metabolism by matching focal increases in neuronal activity with local arteriolar dilation. Previously, we demonstrated that an emergence of spontaneous endfoot high-amplitude Ca2+ signals (eHACSs) caused a pathologic shift in neurovascular coupling from vasodilation to vasoconstriction in brain slices obtained from subarachnoid hemorrhage model animals. Extracellular purine nucleotides (e.g., ATP) can trigger astrocyte Ca2+ oscillations and may be elevated following subarachnoid hemorrhage. Here, the role of purinergic signaling in subarachnoid hemorrhage-induced eHACSs and inversion of neurovascular coupling was examined by imaging parenchymal arteriolar diameter and astrocyte Ca2+ signals in rat brain slices using two-photon fluorescent and infrared-differential interference contrast microscopy. We report that broad-spectrum inhibition of purinergic (P2) receptors using suramin blocked eHACSs and restored vasodilatory neurovascular coupling after subarachnoid hemorrhage. Importantly, eHACSs were also abolished using a cocktail of inhibitors targeting Gq-coupled P2Y receptors. Further, activation of P2Y receptors in brain slices from un-operated animals triggered high-amplitude Ca2+ events resembling eHACSs and disrupted neurovascular coupling. Neither tetrodotoxin nor bafilomycin A1 affected eHACSs suggesting that purine nucleotides are not released by ongoing neurotransmission and/or vesicular release after subarachnoid hemorrhage. These results indicate that purinergic signaling via P2Y receptors contributes to subarachnoid hemorrhage-induced eHACSs and inversion of neurovascular coupling.

Astrocyte Ca2+ Signaling Drives Inversion of Neurovascular Coupling after Subarachnoid Hemorrhage.

Physiologically, neurovascular coupling (NVC) matches focal increases in neuronal activity with local arteriolar dilation. Astrocytes participate in NVC by sensing increased neurotransmission and releasing vasoactive agents (e.g., K(+)) from perivascular endfeet surrounding parenchymal arterioles. Previously, we demonstrated an increase in the amplitude of spontaneous Ca(2+) events in astrocyte endfeet and inversion of NVC from vasodilation to vasoconstriction in brain slices obtained from subarachnoid hemorrhage (SAH) model rats. However, the role of spontaneous astrocyte Ca(2+) signaling in determining the polarity of the NVC response remains unclear. Here, we used two-photon imaging of Fluo-4-loaded rat brain slices to determine whether altered endfoot Ca(2+) signaling underlies SAH-induced inversion of NVC. We report a time-dependent emergence of endfoot high-amplitude Ca(2+) signals (eHACSs) after SAH that were not observed in endfeet from unoperated animals. Furthermore, the percentage of endfeet with eHACSs varied with time and paralleled the development of inversion of NVC. Endfeet with eHACSs were present only around arterioles exhibiting inversion of NVC. Importantly, depletion of intracellular Ca(2+) stores using cyclopiazonic acid abolished SAH-induced eHACSs and restored arteriolar dilation in SAH brain slices to two mediators of NVC (a rise in endfoot Ca(2+) and elevation of extracellular K(+)). These data indicate a causal link between SAH-induced eHACSs and inversion of NVC. Ultrastructural examination using transmission electron microscopy indicated that a similar proportion of endfeet exhibiting eHACSs also exhibited asymmetrical enlargement. Our results demonstrate that subarachnoid blood causes a delayed increase in the amplitude of spontaneous intracellular Ca(2+) release events leading to inversion of NVC. Significance statement: Aneurysmal subarachnoid hemorrhage (SAH)--strokes involving cerebral aneurysm rupture and release of blood onto the brain surface--are associated with high rates of morbidity and mortality. A common complication observed after SAH is the development of delayed cerebral ischemia at sites often remote from the site of rupture. Here, we provide evidence that SAH-induced changes in astrocyte Ca(2+) signaling lead to a switch in the polarity of the neurovascular coupling response from vasodilation to vasoconstriction. Thus, after SAH, signaling events that normally lead to vasodilation and enhanced delivery of blood to active brain regions cause vasoconstriction that would limit cerebral blood flow. These findings identify astrocytes as a key player in SAH-induced decreased cortical blood flow.

SAH-induced MMP activation and K V current suppression is mediated via both ROS-dependent and ROS-independent mechanisms.

Voltage-gated potassium (K V) channels regulate cerebral artery tone and have been implicated in subarachnoid hemorrhage (SAH)-induced pathologies. Here, we examined whether matrix metalloprotease (MMP) activation contributes to SAH-induced K V current suppression and cerebral artery constriction via activation of epidermal growth factor receptors (EGFRs). Using patch clamp electrophysiology, we observed that K V currents were selectively decreased in cerebral artery myocytes isolated from SAH model rabbits. Consistent with involvement of enhanced MMP and EGFR activity in SAH-induced K V current suppression, we found that: (1) oxyhemoglobin (OxyHb) and/or the exogenous EGFR ligand, heparin-binding EGF-like growth factor (HB-EGF), failed to induce further K V current suppression after SAH and (2) gelatin zymography detected significantly higher MMP-2 activity after SAH. The removal of reactive oxygen species (ROS) by combined treatment with superoxide dismutase (SOD) and catalase partially inhibited OxyHb-induced K V current suppression. However, these agents had little effect on OxyHb-induced MMP-2 activation. Interestingly, in the presence of a broad-spectrum MMP inhibitor (GM6001), OxyHb failed to cause K V current suppression. These data suggest that OxyHb suppresses K V currents through both ROS-dependent and ROS-independent pathways involving MMP activation. The ROS-independent pathway involves activation of MMP-2, whereas the ROS-dependent pathway involves activation of a second unidentified MMP or ADAM (a disintegrin and metalloprotease domain).

Activation of TRPV4 channels does not mediate inversion of neurovascular coupling after SAH.

Neurovascular coupling (NVC) allows increased blood flow to metabolically active neurons and involves the Ca²âº -dependent release of vasodilator influences by astrocyte endfeet that encase parenchymal arterioles. We previously reported inversion of NVC from dilation to constriction in brain slices from subarachnoid hemorrhage (SAH) model rats. Corresponding to NVC inversion, there was a marked increase in the amplitude of spontaneous Ca²âº oscillations in astrocyte endfeet. Calcium-permeable transient receptor potential vanilloid (TRPV)-4 channels have been reported in astrocyte endfeet, and activators of these channels enhance Ca²âº oscillations in healthy animals. Here, we examined the role of TRPV4 channels in the development of high-amplitude spontaneous Ca²âº oscillations in astrocyte endfeet and the inversion of neurovascular coupling after SAH. Treatment of brain slices with the TRPV4 channel antagonist, HC-067047 (10 µM), did not alter the amplitude of spontaneous Ca²âº oscillations after SAH. In addition, HC-067047 did not inhibit or change SAH-induced inversion of neurovascular coupling. In summary, TRPV4 channels do not appear to be involved in the inversion of neurovascular coupling after SAH. Further studies examining the impact of SAH on additional Ca²âº signaling pathways in astrocytes are likely to reveal valuable insights into new therapeutic strategies to advance SAH treatments.

Traumatic brain injury disrupts cerebrovascular tone through endothelial inducible nitric oxide synthase expression and nitric oxide gain of function.

BACKGROUND: Traumatic brain injury (TBI) has been reported to increase the concentration of nitric oxide (NO) in the brain and can lead to loss of cerebrovascular tone; however, the sources, amounts, and consequences of excess NO on the cerebral vasculature are unknown. Our objective was to elucidate the mechanism of decreased cerebral artery tone after TBI. METHODS AND RESULTS: Cerebral arteries were isolated from rats 24 hours after moderate fluid­percussion TBI. Pressure­induced increases in vasoconstriction (myogenic tone) and smooth muscle Ca2+ were severely blunted in cerebral arteries after TBI. However, myogenic tone and smooth muscle Ca2+ were restored by inhibition of NO synthesis or endothelium removal, suggesting that TBI increased endothelial NO levels. Live native cell NO, indexed by 4,5­diaminofluorescein (DAF­2 DA) fluorescence, was increased in endothelium and smooth muscle of cerebral arteries after TBI. Clamped concentrations of 20 to 30 nmol/L NO were required to simulate the loss of myogenic tone and increased (DAF­2T) fluorescence observed following TBI. In comparison, basal NO in control arteries was estimated as 0.4 nmol/L. Consistent with TBI causing enhanced NO­mediated vasodilation, inhibitors of guanylyl cyclase, protein kinase G, and large­conductance Ca2+­activated potassium (BK) channel restored function of arteries from animals with TBI. Expression of the inducible isoform of NO synthase was upregulated in cerebral arteries isolated from animals with TBI, and the inducible isoform of NO synthase inhibitor 1400W restored myogenic responses following TBI. CONCLUSIONS: The mechanism of profound cerebral artery vasodilation after TBI is a gain of function in vascular NO production by 60­fold over controls, resulting from upregulation of the inducible isoform of NO synthase in the endothelium.

Evidence against a systemic arterial defect in patients with inflammatory bowel disease.

BACKGROUND: Despite increasing interest in local microvascular alterations associated with inflammatory bowel disease (IBD), the potential contribution of a primary systemic vascular defect in the etiology of IBD is unknown. We compared reactivity of large diameter mesenteric arteries from segments affected by Crohn disease (CD) or ulcerative colitis (UC) to an uninvolved vascular bed in both IBD and control patients. METHODS: Mesenteric and omental arteries were obtained from UC, CD, and non-IBD patients. Isometric arterial contractions were recorded in response to extracellular potassium (K(+)) and cumulative additions of norepinephrine (NE). In addition, relaxation in response to pinacidil, an activator of adenosine triphosphate-sensitive K(+) channels was examined. RESULTS: Contraction to K(+) and sensitivity to NE were not significantly different in arteries from CD, UC, and controls. Relaxation to pinacidil was also similar between groups. CONCLUSIONS: Potassium-induced contractions and sensitivity to NE and pinacidil were not significantly different in large diameter mesenteric and omental arteries obtained from IBD patients. Furthermore, there was no significant difference in the sensitivity to K(+), NE, and pinacidil between mesenteric and omental arteries of CD and UC patients and those from non-IBD patients. Our results suggest an underlying vascular defect systemic to CD or UC patients is unlikely to contribute to the etiology of IBD.

Pituitary adenylate cyclase activating polypeptide (PACAP) dilates cerebellar arteries through activation of large-conductance Ca(2+)-activated (BK) and ATP-sensitive (K ATP) K (+) channels.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a potent vasodilator of numerous vascular beds, including cerebral arteries. Although PACAP-induced cerebral artery dilation is suggested to be cyclic AMP (cAMP)-dependent, the downstream intracellular signaling pathways are still not fully understood. In this study, we examined the role of smooth muscle K(+) channels and hypothesized that PACAP-mediated increases in cAMP levels and protein kinase A (PKA) activity result in the coordinate activation of ATP-sensitive K(+) (KATP) and large-conductance Ca(2+)-activated K(+) (BK) channels for cerebral artery dilation. Using patch-clamp electrophysiology, we observed that PACAP enhanced whole-cell KATP channel activity and transient BK channel currents in freshly isolated rat cerebellar artery myocytes. The increased frequency of transient BK currents following PACAP treatment is indicative of increased intracellular Ca(2+) release events termed Ca(2+) sparks. Consistent with the electrophysiology data, the PACAP-induced vasodilations of cannulated cerebellar artery preparations were attenuated by approximately 50 % in the presence of glibenclamide (a KATP channel blocker) or paxilline (a BK channel blocker). Further, in the presence of both blockers, PACAP failed to cause vasodilation. In conclusion, our results indicate that PACAP causes cerebellar artery dilation through two mechanisms: (1) KATP channel activation and (2) enhanced BK channel activity, likely through increased Ca(2+) spark frequency.