Astrocyte inositol triphosphate receptor type 2 and cytosolic phospholipase a(2) alpha regulate arteriole responses in mouse neocortical brain slices

Functional hyperemia of the cerebral vascular system matches regional blood flow to the metabolic demands of the brain. One current model of neurovascular control holds that glutamate released by neurons activates group I metabotropic glutamate receptors (mGluRs) on astrocytes, resulting in the production of diffusible messengers that act to regulate smooth muscle cells surrounding cerebral arterioles. The acute mouse brain slice is an experimental system in which changes in arteriole diameter can precisely measured with light microscopy. Stimulation of the brain slice triggers specific cellular responses that can be correlated to changes in arteriole diameter. Here we used inositol trisphosphate receptor type 2 (IP(3)R2) and cytosolic phospholipase A(2) alpha (cPLA(2)α) deficient mice to determine if astrocyte mGluR activation coupled to IP(3)R2-mediated Ca(2+) release and subsequent cPLA(2)α activation is required for arteriole regulation. We measured changes in astrocyte cytosolic free Ca(2+) and arteriole diameters in response to mGluR agonist or electrical field stimulation in acute neocortical mouse brain slices maintained in 95% or 20% O(2). Astrocyte Ca(2+) and arteriole responses to mGluR activation were absent in IP(3)R2(-) (/-) slices. Astrocyte Ca(2+) responses to mGluR activation were unchanged by deletion of cPLA(2)α but arteriole responses to either mGluR agonist or electrical stimulation were ablated. The valence of changes in arteriole diameter (dilation/constriction) was dependent upon both stimulus and O(2) concentration. Neuron-derived NO and activation of the group I mGluRs are required for responses to electrical stimulation. These findings indicate that an mGluR/IP(3)R2/cPLA(2)α signaling cascade in astrocytes is required to transduce neuronal glutamate release into arteriole responses.     

High dietary salt reduces the contribution of 20-HETE to arteriolar oxygen responsiveness in skeletal muscle

The coupling of tissue blood flow to cellular metabolic demand involves oxygen-dependent adjustments in arteriolar tone, and arteriolar responses to oxygen can be mediated, in part, by changes in local production of 20-HETE. In this study, we examined the long-term effect of dietary salt on arteriolar oxygen responsiveness in the exteriorized, superfused rat spinotrapezius muscle and the role of 20-HETE in this responsiveness. Rats were fed either a normal-salt (NS, 0.45%) or high-salt (HS, 4%) diet for 4-5 wk. There was no difference in steady-state tissue Po(2) between NS and HS rats, and elevation of superfusate oxygen content from 0% to 10% caused tissue Po(2) to increase by the same amount in both groups. However, the resulting reductions in arteriolar diameter and blood flow were less in HS rats than NS rats. Inhibition of 20-HETE formation with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) or 17-octadecynoic acid (17-ODYA) attenuated oxygen-induced constriction in NS rats but not HS rats. Exogenous 20-HETE elicited arteriolar constriction that was greatly reduced by the large-conductance Ca(2+)-activated potassium (K(Ca)) channel inhibitors tetraethylammonium chloride (TEA) and iberiotoxin (IbTx) in NS rats and a smaller constriction that was less sensitive to TEA or IbTx in HS rats. Arteriolar responses to exogenous angiotensin II were similar in both groups but more sensitive to inhibition with DDMS in NS rats. Norepinephrine-induced arteriolar constriction was similar and insensitive to DDMS in both groups. We conclude that 20-HETE contributes to oxygen-induced constriction of skeletal muscle arterioles via inhibition of K(Ca) channels and that a high-salt diet impairs arteriolar responses to increased oxygen availability due to a reduction in vascular smooth muscle responsiveness to 20-HETE.     

Origins of the BOLD changes due to synaptic activity at astrocytes abutting arteriolar smooth muscle

We have recently provided a detailed model that links glutamatergic synaptic activity to volume and blood flow changes in nearby arterioles [Bennett, M.R., Farnell, L., Gibson, W.G., 2008. Origin of blood volume change due to glutamatergic synaptic activity at astrocytes abutting on arteriolar smooth muscle cells. J. Theor. Biol. 250, 172-185]. This neurovascular coupling model is used in the present work to predict changes in deoxyhemoglobin (Hbr) in capillaries, arterioles, venules and veins due to glutamatergic synaptic activity and hence the changes in the blood oxygen level dependent (BOLD) signals recorded by functional magnetic resonance imaging. The model provides a quantitative account of Hbr changes observed in each of the vascular compartments following stimulation of somatosensory cortex and visual cortex and of the BOLD signal following stimulation of motor and visual cortex.     

Role of astrocytes in cerebrovascular regulation.

Astrocytes send processes to synapses and blood vessels, communicate with other astrocytes through gap junctions and by release of ATP, and thus are an integral component of the neurovascular unit. Electrical field stimulations in brain slices demonstrate an increase in intracellular calcium in astrocyte cell bodies transmitted to perivascular end-feet, followed by a decrease in vascular smooth muscle calcium oscillations and arteriolar dilation. The increase in astrocyte calcium after neuronal activation is mediated, in part, by activation of metabotropic glutamate receptors. Calcium signaling in vitro can also be influenced by adenosine acting on A2B receptors and by epoxyeicosatrienoic acids (EETs) shown to be synthesized in astrocytes. Prostaglandins, EETs, arachidonic acid, and potassium ions are candidate mediators of communication between astrocyte end-feet and vascular smooth muscle. In vivo evidence supports a role for cyclooxygenase-2 metabolites, EETs, adenosine, and neuronally derived nitric oxide in the coupling of increased blood flow to increased neuronal activity. Combined inhibition of the EETs, nitric oxide, and adenosine pathways indicates that signaling is not by parallel, independent pathways. Indirect pharmacological results are consistent with astrocytes acting as intermediaries in neurovascular signaling within the neurovascular unit. For specific stimuli, astrocytes are also capable of transmitting signals to pial arterioles on the brain surface for ensuring adequate inflow pressure to parenchymal feeding arterioles. Therefore, evidence from brain slices and indirect evidence in vivo with pharmacological approaches suggest that astrocytes play a pivotal role in regulating the fundamental physiological response coupling dynamic changes in cerebral blood flow to neuronal synaptic activity. Future work using in vivo imaging and genetic manipulation will be required to provide more direct evidence for a role of astrocytes in neurovascular coupling.     

Perivascular nerves and the regulation of cerebrovascular tone.

Brain perfusion is tightly coupled to neuronal activity, is commonly used to monitor normal or pathological brain function, and is a direct reflection of the interactions that occur between neuronal signals and blood vessels. Cerebral blood vessels at the surface and within the brain are surrounded by nerve fibers that originate, respectively, from peripheral nerve ganglia and intrinsic brain neurons. Although of different origin and targeting distinct vascular beds, these "perivascular nerves" fulfill similar roles related to cerebrovascular functions, a major one being to regulate their tone and, therein, brain perfusion. This utmost function, which underlies the signals used in functional neuroimaging techniques and which can be jeopardized in pathologies such as Alzheimer's disease, stroke, and migraine headache, is thus regulated at several levels. Recently, new insights into our understanding of how neural input regulate cerebrovascular tone resulted in the rediscovery of the functional "neurovascular unit." These remarkable advances suggest that neuron-driven changes in vascular tone result from interactions that involve all components of the neurovascular unit, transducing neuronal signals into vasomotor responses not only through direct interaction between neurons and vessels but also indirectly via the perivascular astrocytes. Neurovascular coupling is thus determined by chemical signals released from activated perivascular nerves and astrocytes that alter vascular tone to locally adjust perfusion to the spatial and temporal changes in brain activity.     

Evidence for impaired neurovascular transmission in a murine model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a muscle-wasting disease caused by mutations in the dystrophin gene. Little is known about how blood flow control is affected in arteriolar networks supplying dystrophic muscle. We tested the hypothesis that mdx mice, a murine model for DMD, exhibit defects in arteriolar vasomotor control. The cremaster muscle was prepared for intravital microscopy in pentobarbital sodium-anesthetized mdx and C57BL/10 control mice (n ≥ 5 per group). Spontaneous vasomotor tone increased similarly with arteriolar branch order in both mdx and C57BL/10 mice [pooled values: first order (1A), 6%; second order (2A), 56%; and third order (3A), 61%] with no difference in maximal diameters between groups measured during equilibration with topical 10 μM sodium nitroprusside (pooled values: 1A, 70 ± 3 μm; 2A, 31 ± 3 μm; and 3A, 19 ± 3 μm). Concentration-response curves to acetylcholine (ACh) and norepinephrine added to the superfusion solution did not differ between mdx and C57BL/10 mice, nor did constriction to elevated (21%) oxygen. In response to local stimulation from a micropipette, conducted vasodilation to ACh and conducted vasoconstriction to KCl were also not different between groups; however, constriction decayed with distance (P < 0.05) whereas dilation did not. Remarkably, arteriolar constriction to perivascular nerve stimulation (PNS) at 2, 4, and 8 Hz was reduced by ～25-30% in mdx mice compared with C57BL/10 mice (P < 0.05). With intact arteriolar reactivity to agonists, attenuated constriction to perivascular nerve stimulation indicates impaired neurovascular transmission in arterioles controlling blood flow in mdx mice.     

Adenosine linking reduced O2 to arteriolar NO release in intestine is not formed from extracellular ATP

We have previously reported that adenosine formed in response to reduced arteriolar and/or tissue PO(2) preserves endothelial nitric oxide (NO) synthesis during sympathetic vasoconstriction in the rat intestine. To more precisely identify the site and mechanism of adenosine formation under these conditions, we tested the hypothesis that ATP released in response to reduced O(2) levels serves as a source of adenosine. Direct application of ATP to the wall of first-order arterioles elicited dose-dependent dilations of 15-33% above resting diameter that were reduced by 71-80% by the 5'-ectonucleotidase inhibitor alpha,beta-methyleneadenosine 5'-diphosphate (AOPCP, 4.5 x 10(-5) M) and completely abolished by N(G)-monomethyl-L-arginine (L-NMMA, 10(-4) M). Under control conditions, sympathetic nerve stimulation at 3 and 8 Hz induced arteriolar constrictions of 11 +/- 1 and 19 +/- 1 microm, respectively. These responses were enhanced by 58-69% in the presence of L-NMMA or when local PO(2) was maintained at resting levels. However, in the presence of AOPCP, the enhancing effects of L-NMMA and the high O(2) superfusate on sympathetic constriction were preserved. These results suggest that, although exogenously applied ATP can stimulate arteriolar NO release in the intestine largely through its sequential extracellular hydrolysis to adenosine, this process does not contribute to adenosine formation and sustained NO release during sympathetic constriction in this vascular bed.     

Tyrosine phosphorylation modulates arteriolar tone but is not fundamental to myogenic response

The present study investigated the role of protein tyrosine phosphorylation in myogenic responsiveness of rat skeletal muscle arterioles. Arteriolar segments were cannulated and pressurized without intraluminal flow. All vessels studied developed spontaneous tone and demonstrated significant myogenic constriction to step changes in pressure with a resultant increase in myogenic tone over an intraluminal pressure range of 50-150 mmHg. Step increases in intraluminal pressure from 50 to 120 mmHg caused a rapid and sustained elevation in intracellular [Ca(2+)], as measured using fura 2. Vessels with myogenic tone dilated in response to tyrosine kinase inhibitors genistein (10 or 30 microM) and tyrphostin A47 (10 or 30 microM) and constricted to the tyrosine phosphatase inhibitor pervanadate (1 or 10 microM). Despite the dilator effect, myogenic reactivity was not blocked by the inhibitors. Daidzein (10 microM), a compound structurally similar to genistein but without tyrosine kinase-inhibiting activity, did not alter vessel tone or myogenic responses. Preincubation of arterioles with genistein or tyrphostin A47 did not significantly alter baseline arteriolar [Ca(2+)], and neither drug reduced the increase in [Ca(2+)] following an acute increase in intraluminal pressure. Constriction induced by pervanadate (10 microM) was not accompanied by a significant increase in intracellular [Ca(2+)], even though removal of extracellular Ca(2+) reversed the constriction. Examination of smooth muscle tyrosine phosphorylation, using a fluorescent phosphotyrosine antibody and confocal microscopy, showed that increased intraluminal pressure resulted in an increase in anti-phosphotyrosine fluorescence. Because manipulation of tyrosine kinase activity was found to alter vessel diameter, these data support a role for tyrosine phosphorylation in modulation of arteriolar tone. However, the results indicate that acute arteriolar myogenic constriction does not require tyrosine phosphorylation.     

Epoxyeicosatrienoic acid-dependent cerebral vasodilation evoked by metabotropic glutamate receptor activation in vivo

Group I metabotropic glutamate receptors (mGluR) on astrocytes have been shown to participate in cerebral vasodilation to neuronal activation in brain slices. Pharmacological stimulation of mGluR in brain slices can produce arteriolar constriction or dilation depending on the initial degree of vascular tone. Here, we examined whether pharmacological stimulation of mGluR in vivo increases cerebral blood flow. A 1-mM solution of the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) superfused at 5 μl/min over the cortical surface of anesthetized rats produced a 30 ± 2% (±SE) increase in blood flow measured by laser-Doppler flowmetry after 15-20 min. The response was completely blocked by superfusion of group I mGluR antagonists and attenuated by superfusion of an epoxyeicosatrienoic acid (EET) antagonist (5 ± 4%), an EET synthesis inhibitor (11 ± 3%), and a cyclooxygenase-2 inhibitor (15 ± 3%). The peak blood flow response was not significantly affected by administration of inhibitors of cyclooxygenase-1, neuronal nitric oxide synthase, heme oxygenase, adenosine A(2B) receptors, or an inhibitor of the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE). The blood flow response gradually waned following 30-60 min of DHPG superfusion. This loss of the flow response was attenuated by a 20-HETE synthesis inhibitor and was prevented by superfusion of an inhibitor of epoxide hydrolase, which hydrolyzes EETs. These results indicate that pharmacological stimulation of mGluR in vivo increases cerebral blood flow and that the response depends on the release of EETs and a metabolite of cyclooxygenase-2. Epoxide hydrolase activity and 20-HETE synthesis limit the duration of the response to prolonged mGluR activation.     

Cortical spreading depression confounds concentration-dependent pial arteriolar dilation during N-methyl-D-aspartate superfusion

Pial arterioles do not express N-methyl-D-aspartate (NMDA) receptors but dilate in response to topical NMDA application. We explored the mechanism underlying NMDA-mediated responses in murine pial arterioles (11-31 microm), using a closed cranial window preparation, and found that arteriolar dilation was not concentration dependent. Pial arteriolar diameter abruptly increased within 3 min of superfusing 50 or 100 microM NMDA. Dilation reached a peak within 1 min (46 +/- 14%) and then declined to a plateau (28 +/- 13%) for the duration of superfusion. Whereas a higher concentration (200 microM) did not produce further dilation, lower concentrations (1-10 microM) did not dilate the arterioles at all. MK-801 (10 microM) abrogated the dilation response, whereas Nomega-nitro-L-arginine (1 mM) attenuated the peak and abolished the sustained dilation during NMDA superfusion. We determined that NMDA-induced pial arteriolar responses were evoked by cortical spreading depression, because abrupt vasodilation during 50 or 100 microM NMDA superfusion was associated with a large negative slow potential shift and electrocorticogram suppression that spread from the superfusion window to distant cortical areas. Our data suggest that the responses of pial arterioles to NMDA are caused in part by neurovascular coupling due to cortical spreading depression.     

Transfection of CYP4A1 cDNA decreases diameter and increases responsiveness of gracilis muscle arterioles to constrictor stimuli

Cytochrome P-450-4A1 (CYP4A1) is an omega-hydroxylase that catalyzes the metabolism of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE). The goal of this study was to determine the vasomotor consequences of vascular overexpression of CYP4A1. Isolated rat gracilis muscle arterioles transfected ex vivo with an expression plasmid containing CYP4A1 cDNA expressed more CYP4A protein than vessels transfected with the control plasmid. In arterioles pressurized to 80 mmHg, the internal diameter of vessels transfected with CYP4A1 cDNA (55 +/- 3 microm) was surpassed (P < 0.05) by that of vessels transfected with control plasmid (97 +/- 4 microm). Treatment with a CYP4A inhibitor (N-methylsulfonyl-12,12-dibromododec-11-enamide; DDMS) or with an antagonist of 20-HETE actions [20-hydroxyeicosa-6(Z),15(Z)-dienoic acid; 20-HEDE] elicited robust dilation of arterioles transfected with CYP4A1 cDNA, whereas the treatment had little or no effect in vessels transfected with control plasmid. Examination of the intraluminal pressure-internal diameter relationship revealed that pressure increments over the range of 40-100 mmHg elicited a more intense (P < 0.05) myogenic constrictor response in arterioles transfected with CYP4A1 cDNA than in those with control plasmid. Arterioles transfected with CYP4A1 cDNA also displayed enhanced sensitivity to the constrictor action of phenylephrine. Treatment with DDMS or 20-HEDE greatly attenuated the constrictor responsiveness to both constrictor stimuli in vessels overexpressing CYP4A1, whereas the treatment had much less effect in control vessels. These data suggest that CYP4A1 overexpression promotes constriction of gracilis muscle arterioles by intensifying the responsiveness of vascular smooth muscle to constrictor stimuli. This effect of CYP4A1 overexpression appears to be mediated by a CYP4A1 product.     

Astrocytes are a key conduit for upstream signaling of vasodilation during cerebral cortical neuronal activation in vivo

Astrocytes play an important role in the coupling between neuronal activity and brain blood flow via their capacity to "sense" neuronal activity and transmit that information to parenchymal arterioles. Here we show another role for astrocytes in neurovascular coupling: the ability to act as a signaling conduit for the vitally important process of upstream vasodilation (represented by pial arterioles) during both excessive (seizure) and physiological (sciatic nerve stimulation) increases in cerebral cortical neuronal activity. The predominance of an astrocytic rather than a vascular route was indicated by data showing that pial arteriolar-dilating responses to neuronal activation were completely blocked following selective disruption of the superficial glia limitans, whereas interference with interendothelial signaling was without effect. Results also revealed contributions from connexin 43, implying a role for gap junctions and/or hemichannels in the signaling process and that signaling from the glia limitans to pial arterioles may involve a diffusible mediator.     

Potassium channels modulate cerebral autoregulation during acute hypertension

We tested the hypothesis that constriction of cerebral arterioles during acute increases in blood pressure is attenuated by activation of potassium (K(+)) channels. We tested the effects of inhibitors of calcium-dependent K(+) channels [iberiotoxin (50 nM) and tetraethylammonium (TEA, 1 mM)] on changes in arteriolar diameter during acute hypertension. Diameter of cerebral arterioles (baseline diameter = 46 +/- 2 microm, mean +/- SE) was measured using a cranial window in anesthetized rats. Arterial pressure was increased from a control value of 96 +/- 1 mmHg to 130, 150, 170, and 200 mmHg by intravenous infusion of phenylephrine. Increases in arterial pressure from baseline to 130 and 150 mmHg decreased the diameter of cerebral arterioles by 5-10%. Greater increases in arterial pressure produced large increases in arteriolar diameter (i.e., "breakthrough of autoregulation"). Iberiotoxin or TEA inhibited increases in arteriolar diameter when arterial pressure was increased to 170 and 200 mmHg. The change in arteriolar diameter at 200 mmHg was 20 +/- 3% and -1 +/- 4% in the absence and presence of iberiotoxin, respectively. These findings suggest that calcium-dependent K(+) channels attenuate cerebral microvascular constriction during acute increases in arterial pressure, and that increases in arteriolar diameter at high levels of arterial pressure are not simply a passive phenomenon.     

Impairment of neurovascular coupling in type 1 diabetes mellitus in rats is linked to PKC modulation of BK(Ca) and Kir channels

We hypothesized that chronic hyperglycemia has a detrimental effect on neurovascular coupling in the brain and that this may be linked to protein kinase C (PKC)-mediated phosphorylation. Therefore, in a rat model of streptozotocin-induced chronic type 1 diabetes mellitus (T1DM), and in nondiabetic (ND) controls, we monitored pial arteriole diameter changes during sciatic nerve stimulation and topical applications of the large-conductance Ca(2+)-operated K(+) channel (BK(Ca)) opener, NS-1619, or the K(+) inward rectifier (Kir) channel agonist, K(+). In the T1DM vs. ND rats, the dilatory response associated with sciatic nerve stimulation was decreased by ～30%, whereas pial arteriolar dilations to NS-1619 and K(+) were largely suppressed. These responses were completely restored by the acute topical application of a PKC antagonist, calphostin C. Moreover, the suffusion of a PKC activator, phorbol 12,13-dibutyrate, in ND rats was able to reproduce the vascular reactivity impairments found in T1DM rats. Assay of PKC activity in brain samples from T1DM vs. ND rats revealed a significant gain in activity only in specimens harvested from the pial and superficial glia limitans tissue, but not in bulk cortical gray matter. Altogether, these findings suggest that the T1DM-associated impairment of neurovascular coupling may be mechanistically linked to a readily reversible PKC-mediated depression of BK(Ca) and Kir channel activity.     

Contribution of cytochrome P-450 omega-hydroxylase to altered arteriolar reactivity with high-salt diet and hypertension

The present study evaluated the contribution of cytochrome P-450 omega-hydroxylase in modulating the reactivity of cremaster muscle arterioles in normotensive rats on high-salt (HS) and low-salt (LS) diet and in rats with reduced renal mass hypertension (RRM-HT). Changes in arteriolar diameter in response to ACh, sodium nitroprusside (SNP), ANG II, and elevated O(2) were measured via television microscopy under control conditions and following cytochrome P-450 omega-hydroxylase inhibition with 17-octadecynoic acid (17-ODYA) or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS). In normotensive rats on either LS or HS diet, resting tone was unaffected and arteriolar reactivity to ACh or SNP was minimally affected by cytochrome P-450 omega-hydroxylase inhibition. In RRM-HT rats, cytochrome P-450 omega-hydroxylase inhibition reduced resting tone and significantly enhanced arteriolar dilation to ACh and SNP. Treatment with 17-ODYA or DDMS inhibited arteriolar constriction to ANG II and O(2) in all the groups, although the degree of inhibition was greater in RRM-HT than in normotensive animals. These results suggest that metabolites of cytochrome P-450 omega-hydroxylase contribute to the altered reactivity of skeletal muscle arterioles to vasoconstrictor and vasodilator stimuli in RRM-HT.     

H2O2 increases production of constrictor prostaglandins in smooth muscle leading to enhanced arteriolar tone in Type 2 diabetic mice

Our previous study showed that arteriolar tone is enhanced in Type 2 diabetes mellitus (T2-DM) due to an increased level of constrictor prostaglandins. We hypothesized that, in mice with T2-DM, hydrogen peroxide (H(2)O(2)) is involved in the increased synthesis of constrictor prostaglandins, hence enhanced basal tone in skeletal muscle arterioles. Isolated, pressurized gracilis muscle arterioles ( approximately 100 microm in diameter) of mice with T2-DM (C57BL/KsJ-db(-)/db(-)) exhibited greater basal tone to increases in intraluminal pressure (20-120 mmHg) than that of control vessels (at 80 mmHg, control: 25 +/- 5%; db/db: 34 +/- 4%, P < 0.05), which was reduced back to control level by catalase (db/db: 24 +/- 4%). Correspondingly, in carotid arteries of db/db mice, the level of dichlorofluorescein-detectable and catalase-sensitive H(2)O(2) was significantly greater. In control arterioles, exogenous H(2)O(2) (0.1-100 micromol/l) elicited dilations (maximum, 58 +/- 10%), whereas in arterioles of db/db mice H(2)O(2) caused constrictions (-28 +/- 8%), which were converted to dilations (maximum, 16 +/- 5%) by the thromboxane A(2)/prostaglandin H(2) (TP) receptor antagonist SQ-29548. In addition, arteriolar constrictions in response to the TP receptor agonist U-46619 were not different between the two groups of vessels. Endothelium denudation did not significantly affect basal tone and H(2)O(2)-induced arteriolar responses in either control or db/db mice. Also, in arterioles of db/db mice, but not in controls, 3-nitrotyrosine staining was detected in the endothelial layer of vessels. Thus we propose that, in mice with T2-DM, arteriolar production of H(2)O(2) is enhanced, which leads to increased synthesis of the constrictor prostaglandins thromboxane A(2)/prostaglandin H(2) in the smooth muscle cells, which enhance basal arteriolar tone. These alterations may contribute to disturbed regulation of skeletal muscle blood flow in Type 2 diabetes mellitus.     

Conducted dilations initiated by purines in arterioles are endothelium dependent and require endothelial Ca2+

The signaling pathways underlying the regulation of vascular resistance by purines in intact microvessels and particularly in communication of remote vasomotor responses are unclear. One process by which remote regions of arterioles communicate is via transmission of signals axially along the vessel wall. In this study, we identified a pathway for local and conducted dilations initiated by purines. Adenosine (Ado) or ATP (bind P1 and P2 purinergic receptors, respectively) was micropipette applied to arterioles (maximum diameter approximately 40 microm) in the cheek pouch of anesthetized hamsters. Observations were made at the site of stimulation (local) or approximately 1200 microm upstream along the same vessel. P2 antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium and suramin) inhibited local constriction to ATP, whereas local and upstream dilations were unaffected. In contrast, during inhibition of P1 receptors (with xanthine amine congener) the local constriction was unchanged, whereas both local and upstream dilations to ATP were inhibited. Hydrolysis of ATP to Ado is implicated in the dilator response as blocking 5'-ectonucleotidase (with alpha,beta-methyleneadenosine 5'-diphosphate) attenuated ATP-induced dilations. After endothelium denudation, constriction to ATP was unchanged, but dilations to both ATP and Ado were inhibited, identifying endothelial cells (ECs) as the primary target for P1-mediated dilation. Purines increased EC Ca2+ locally and upstream. Chelation of EC Ca2+ (with BAPTA) abolished the local and upstream dilations to P1 receptor stimulation. Collectively, these data demonstrate that stimulation of P1 receptors on ECs produces a vasodilation that spreads to remote regions. There is an associated increase in EC Ca2+, which is a required signaling intermediate in the manifestation of both the local and axially communicated arteriolar dilations.     

Ascorbate protects against impaired arteriolar constriction in sepsis by inhibiting inducible nitric oxide synthase expression

Compromised microvascular responsiveness is one of the key factors associated with mortality of septic patients. The present study addresses the mechanism of protection by ascorbate against impaired vasoconstriction in septic mice. Sepsis (i.e., cecal ligation and puncture (CLP) model) elevated both plasma protein carbonyl (i.e., an index of oxidative stress) and plasma nitrite/nitrate (NOx) levels, reduced baseline mean arterial blood pressure (MABP), and inhibited the MABP pressor response to angiotensin II (Ang II) at 6 h post-CLP. At the microvascular level, sepsis increased the inducible nitric oxide synthase (iNOS) mRNA level in cremaster muscle arterioles (18-25 microm diameter) at 3 h post-CLP, and impaired vasoconstriction to Ang II in these arterioles at 6 h post-CLP. At 24 h post-CLP, sepsis resulted in 9% survival. An intravenous bolus of ascorbate (200 mg/kg body wt) given 30 min prior to CLP prevented the protein carbonyl and NOx increases, partially restored the baseline arterial pressure, and completely protected against all arteriolar iNOS mRNA increases, arteriolar constriction hyporesponsiveness, and pressor response impairment. Survival increased to 65%. In septic mice, iNOS gene knockout resulted in protection of arteriolar constriction and pressor responses identical to that provided by ascorbate. Ascorbate bolus given 3 h post-CLP protected against the increase in plasma NOx concentration and against the pressor response impairment. We conclude that ascorbate may protect arteriolar vasoconstrictor responsiveness in sepsis by inhibiting excessive NO production.     

The effect of PGF2 alpha on in vivo cerebral arteriolar diameter in cats and rats

We compared the effect of topical application of PGF2 alpha on cerebral arterioles in cats and rats equipped with an acutely implanted cranial window. Arterial diameter was measured using a microscope and image splitting device. PGF2 alpha in a concentration ranging from 10(-7) to 10(-5) M had no effect on large (greater than or equal to 100 microns) or small (less than 100 microns) cat pial arterioles, but induced a dose dependent constriction of rat pial arterioles with a maximum constriction to 76% of control diameter. Dilation of cat large cerebral arterioles by topically applied PGE2 was not affected by simultaneous application of PGF2 alpha and PGE2 induced dilation of small arterioles was decreased 3% by PGF2 alpha. While we and others have previously shown that both cat and rat brain can synthesize PGF2 alpha, it appears that PGF2 alpha is not likely to normally be a major modulator of cerebral arteriolar resistance in all species.     

Estimating oxygen consumption rates of arteriolar walls under physiological conditions in rat skeletal muscle

To examine the effects of vascular tone reduction on O2 consumption of the vascular wall, we determined the O2 consumption rates of arteriolar walls under normal conditions and during vasodilation induced by topical application of papaverine. A phosphorescence quenching technique was used to quantify intra- and perivascular PO2 in rat cremaster arterioles with different branching orders. Then, the measured radial PO2 gradients and a theoretical model were used to estimate the O2 consumption rates of the arteriolar walls. The vascular O2 consumption rates of functional arterioles were >100 times greater than those observed in in vitro experiments. The vascular O2 consumption rate was highest in first-order (1A) arterioles, which are located upstream, and sequentially decreased downstream in 2A and 3A arterioles under normal conditions. During papaverine-induced vasodilation, on the other hand, the O2 consumption rates of the vascular walls decreased to similar levels, suggesting that the high O2 consumption rates of 1A arterioles under normal conditions depend in part on the workload of the vascular smooth muscle. These results strongly support the hypothesis that arteriolar walls consume a significant amount of O2 compared with the surrounding tissue. Furthermore, the reduction of vascular tone of arteriolar walls may facilitate an efficient supply of O2 to the surrounding tissue.