Receptor subtypes mediating adenosine-induced dilation of cerebral arterioles

The purpose of this study was to investigate the receptor subtypes that mediate the dilation of rat intracerebral arterioles elicited by adenosine. Penetrating arterioles were isolated from the rat brain, cannulated with the use of a micropipette system, and luminally pressurized to 60 mmHg. Both adenosine and the A2A receptor-selective agonist CGS-21680 induced dose-dependent vasodilation (-logEC(50): 6.5 +/- 0.2 and 8.6 +/- 0.3, respectively). However, adenosine, which is capable of activating both A2A and A2B receptors, caused a greater maximal dilation than CGS-21680. The A2A receptor-selective antagonist ZM-241385 (0.1 microM) only partially inhibited the dilation induced by adenosine but almost completely blocked CGS-21680-induced dilation. Neither 8-cyclopentyl-1,3-dipropylxanthine (0.1 microM), an A1 receptor-selective antagonist, nor MRS-1191 (0.1 microM), an A3 receptor-selective antagonist, attenuated adenosine dose responses. Moreover, ZM-241385 had no effect on the dilation induced by ATP (10 microM) or acidic (pH 6.8) buffer. We concluded that the A2A receptor subtype mediates adenosine-induced dilation of intracerebral arterioles in the rat brain. Furthermore, our results suggest that A2B receptors may also participate in the dilation response to adenosine.     

Contribution of adenosine A2A and A2B receptors and heme oxygenase to AMPA-induced dilation of pial arterioles in rats

Nitric oxide (NO) has been implicated in mediation of cerebral vasodilation during neuronal activation and, specifically, in pharmacological activation of N-methyl-d-aspartate (NMDA) and kainate receptors. Possible mediators of cerebral vasodilation to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) have not been well studied in mature brain, although heme oxygenase (HO) activity has been implicated in newborn pigs. In anesthetized rats, 5 min of topical superfusion of 30 and 100 microM AMPA on the cortical surface through a closed cranial window resulted in increases in pial arteriolar diameter. The dilatory response to AMPA was not inhibited by superfusion of an NO synthase inhibitor, a cyclooxygenase-2 inhibitor, or a cytochrome P-450 epoxygenase inhibitor, all of which have been shown to inhibit the cortical blood flow response to sensory activation. However, the 48 +/- 13% dilation to 100 microM AMPA was attenuated 56-71% by superfusion of the adenosine A(2A) receptor antagonist ZM-241385, the A(2B) receptor antagonist alloxazine, and the HO inhibitor chromium mesoporphyrin. Combination of the latter three inhibitors did not attenuate the dilator response more than the individual inhibitors, whereas an AMPA receptor antagonist fully blocked the vasodilation to AMPA. These results indicate that cortical pial arteriolar dilation to AMPA does not require activation of NO synthase, cyclooxygenase-2, or cytochrome P-450 epoxygenase but does depend on activation of adenosine A(2A) and A(2B) receptors. In addition, CO derived from HO appears to play a role in the vascular response to AMPA receptor activation in mature brain by a mechanism that is not additive with that of adenosine receptor activation.     

Implication of adenosine A2A receptors in hypotension-induced vasodilation and cerebral blood flow autoregulation in rat pial arteries

This study aimed to evaluate the role for adenosine A2A receptors in the autoregulatory vasodilation to hypotension in relation with cerebral blood flow (CBF) autoregulation in rat pial arteries. Changes in pial artery diameters were observed directly through a closed cranial window. Vasodilation induced by adenosine was markedly suppressed by ZM 241385 (1 micromol/l, A2A antagonist) and alloxazine (1 micromol/l, A2B antagonist), but not by 8-cyclopentyltheophylline (CPT, 1 micromol/l, A1 antagonist). CGS-21680-induced vasodilation was more strongly inhibited by ZM 241385 (25.3-fold; P<0.05) than by alloxazine. In contrast, 5'-N-ethylcarboxamido-adenosine (NECA)-induced vasodilation was more prominently suppressed by alloxazine (12.0-fold; P<0.001) than by ZM 241385. The autoregulatory vasodilation in response to acute hypotension of the pial arteries was significantly suppressed by ZM 241385, but not by CPT and alloxazine. Consistent with this finding, the lower limit of CBF autoregulation significantly shifted to a higher blood pressure by 1 micromol/l of ZM 241385 (53.0+/-3.9 mm Hg to 69.2+/-2.9 mm Hg, P<0.01) and 10 micromol/l of glibenclamide (54.7+/-6.5 mm Hg to 77.9+/-4.2 mm Hg, P<0.001), but not by CPT and alloxazine. Thus, it is suggested that adenosine-induced vasodilation of the rat pial artery is mediated via activation of adenosine A2A and A2B receptors, but not by A1 subtype, and activation of adenosine A2A receptor preferentially contributes to the autoregulatory vasodilation via activation of ATP-sensitive K+ channels in response to hypotension and maintenance of CBF autoregulation.     

Functional role of astrocyte glutamate receptors and carbon monoxide in cerebral vasodilation response to glutamate

In newborn pigs, vasodilation of pial arterioles in response to glutamate is mediated via carbon monoxide (CO), a gaseous messenger endogenously produced from heme degradation by a heme oxygenase (HO)-catalyzed reaction. We addressed the hypothesis that ionotropic glutamate receptors (iGluRs), including N-methyl-D-aspartic acid (NMDA)- and 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) propanoic acid (AMPA)/kainate-type receptors, expressed in cortical astrocytes mediate glutamate-induced astrocyte HO activation that leads to cerebral vasodilation. Acute vasoactive effects of topical iGluR agonists were determined by intravital microscopy using closed cranial windows in anesthetized newborn pigs. iGluR agonists, including NMDA, (±)1-aminocyclopentane-cis-1,3-dicarboxylic acid (cis-ACPD), AMPA, and kainate, produced pial arteriolar dilation. Topical L-2-aminoadipic acid, a gliotoxin that selectively disrupts glia limitans, reduced vasodilation caused by iGluR agonists, but not by hypercapnia, bradykinin, or sodium nitroprusside. In freshly isolated and cultured cortical astrocytes constitutively expressing HO-2, iGluR agonists NMDA, cis-ACPD, AMPA, and kainate rapidly increased CO production two- to threefold. Astrocytes overexpressing inducible HO-1 had high baseline CO but were less sensitive to glutamate stimulation of CO production when compared with HO-2-expressing astrocytes. Glutamate-induced astrocyte HO-2-mediated CO production was inhibited by either the NMDA receptor antagonist (R)-3C4HPG or the AMPA/kainate receptor antagonist DNQX. Accordingly, either antagonist abolished pial arteriolar dilation in response to glutamate, NMDA, and AMPA, indicating functional interaction among various subtypes of astrocytic iGluRs in response to glutamate stimulation. Overall, these data indicate that the astrocyte component of the neurovascular unit is responsible for the vasodilation response of pial arterioles to topically applied glutamate via iGluRs that are functionally linked to activation of constitutive HO in newborn piglets.     

Role of adenosine A(2B) receptors in vasodilation of rat pial artery and cerebral blood flow autoregulation

This study was aimed to investigate the underlying mechanism of vasodilation induced by the activation of A(2B) adenosine receptors in relation to cerebral blood flow (CBF) autoregulation. Changes in pial arterial diameters were observed directly through a closed cranial window. N(omega)-nitro-L-arginine methyl ester (L-NAME, nitric oxide synthase inhibitor) significantly suppressed the concentration-dependent vasodilations induced by adenosine and 5'-N-ethylcarboxamido-adenosine (NECA) but not the vasodilation by CGS-21680 (A(2A)-receptor agonist). Moreover, NECA-induced vasodilation was suppressed by alloxazine (1 micromol/l) but not by ZM-241385 (1 micromol/l, A(2A) antagonist), which suggests mediation by A(2B)- receptor activation. Otherwise, the level of nitrite/nitrate was concentration dependently increased in the artificial cerebrospinal fluid (CSF) when adenosine and NECA were suffused over the cortical surface. L-NAME and alloxazine, but not ZM-241385, largely inhibited their releases. The lower limit of CBF autoregulation was little affected following pretreatment with L-NAME or alloxazine. Thus it is suggested that adenosine-induced vasodilation via activation of A(2B)-adenosine receptors of the rat pial artery is coupled to the production of nitric oxide, which contributes little to CBF autoregulation.     

Role of carbon monoxide in glutamate receptor-induced dilation of newborn pig pial arterioles

The hypothesis that glutamate dilates pial arterioles of newborn pigs through the production of carbon monoxide (CO) was addressed. Anesthesized newborn pigs were equipped with cranial windows to measure pial arteriolar responses to stimuli. Heme oxygenase (HO) inhibitors added topically inhibited dilation to glutamate and to specific glutamate receptor agonists. The initial dilation to glutamate (10(-5) M) was 22% from baseline without an inhibitor and decreased to 9% with the HO inhibitor chromium mesoporphyrin (CrMP). Inhibition of dilation upon HO inhibition was similar when specific glutamate receptor agonists were employed. RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid caused 24% dilation from the baseline without an inhibitor, and the dilation was decreased to 1% with tin protoporphyrin (SnPP). (RS)-2-amino-3-(3-hydroxy-5-t-butylisoxazol-4-yl)propionic acid (kainate receptors) caused dilation of 18% from baseline without an inhibitor, but only 2% when tin mesoporphyrin was applied. 1-Aminocyclopropanecarboxylic acid (N-methyl-D-aspartate receptors) dilated pial arterioles 33% from baseline in control, but only to 2% in the presence of SnPP. Neither copper mesoporphyrin, which does not inhibit HO, nor light-inactivated CrMP affected the dilations. Furthermore, cerebral microvessels removed from the brain produced CO (stable isotope dilution gas chromatography-mass spectrometry), and this production was dose dependently increased by glutamate and inhibited by metal porphyrin HO inhibitors. These data suggest that dilation of newborn pig pial arterioles to glutamate and specific glutamate receptor agonists involves vascular production of CO. Additional cerebral sources of CO also could be stimulated by glutamate and contribute to the dilation.     

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.     

Multiple dilator pathways in skeletal muscle contraction-induced arteriolar dilations

To determine whether nitric oxide (NO), adenosine (Ado) receptors, or ATP-sensitive potassium (K(ATP)) channels play a role in arteriolar dilations induced by muscle contraction, we used a cremaster preparation in anesthetized hamsters in which we stimulated four to five muscle fibers lying perpendicular to a transverse arteriole (maximal diameter approximately 35-65 microm). The diameter of the arteriole at the site of overlap of the stimulated muscle fibers (the local site) and at a remote site approximately 1,000 microm upstream (the upstream site) was measured before, during, and after muscle contraction. Two minutes of 4-Hz muscle stimulation (5-15 V, 0.4 ms) produced local and upstream dilations of 19 +/- 1 and 10 +/- 1 microm, respectively. N(omega)-nitro-L-arginine (10(-4) M; NO synthase inhibitor), xanthine amine congener (XAC; 10(-6) M; Ado A(1), A(2A), and A(2B) receptor antagonist), or glibenclamide (Glib; 10(-5) M; K(ATP) channel inhibitor) superfused over the preparation attenuated the local dilation (by 29.7 +/- 12.7, 61.8 +/- 9.0, and 51.9 +/- 14.9%, respectively), but only XAC and Glib attenuated the upstream dilation (by 68.9 +/- 6.8 and 89.1 +/- 6.4%, respectively). Furthermore, only Glib, when applied to the upstream site directly, attenuated the upstream dilation (48.1 +/- 9.1%). Neither XAC nor Glib applied directly to the arteriole between the local and the upstream sites had an effect on the magnitude of the upstream dilation. We conclude that NO, Ado receptors, and K(ATP) channels are involved in the local dilation initiated by contracting muscle and that both K(ATP) channels and Ado receptor stimulation, but not NO, play a role in the manifestation of the dilation at the upstream site.     

Adenosine A(1) and A(2A) receptors modulate sleep state and breathing in fetal sheep.

This study was designed to determine the adenosine (Ado) receptor subtype that mediates the depressant effects of Ado on fetal breathing and rapid eye movements (REM). In chronically catheterized fetal sheep (>0.8 term), intra-arterial infusion of N(6)-cyclopentyladenosine (CPA), an Ado A(1)-receptor agonist, increased the incidence of high-voltage electrocortical (ECoG) activity while virtually abolishing low-voltage activity, REM, and breathing. These effects were blocked by 9-cyclopentyl-1,3-dipropylxanthine (DPCPX), an Ado A(1)-receptor antagonist. Infusion of DPCPX alone increased breath amplitude but had no significant effect on inspiratory duration, breath interval, incidence of REM, or incidence of low-voltage activity. Ado A(2A)-receptor blockade with ZM-241385 increased the incidence of low-voltage ECoG activity, REM, and breathing but had no effect on breath amplitude or respiratory cycle. Both DPCPX and ZM-241385 eliminated the inhibitory effects of Ado on REM and breathing. We conclude that 1) Ado A(1) receptors tonically inhibit fetal respiratory drive, 2) Ado A(2A) receptors tonically inhibit REM-like behavioral state, and 3) both Ado A(1) and A(2A) receptors mediate the depressant effects of Ado on REM and breathing.     

Interactions between adenosine and K+ channel-related pathways in the coupling of somatosensory activation and pial arteriolar dilation

Multiple, perhaps interactive, mechanisms participate in the linkage between increased neural activity and cerebral vasodilation. In the present study, we assessed whether neural activation-related pial arteriolar dilation (PAD) involved interactions among adenosine (Ado) A(2) receptors (A(2)Rs), large-conductance Ca(2+)-operated K(+) (BK(Ca)) channels, and inward rectifier K(+) (K(ir)) channels. In rats with closed cranial windows, we monitored sciatic nerve stimulation (SNS)-induced PAD in the absence or presence of pharmacological blockade of A(2)Rs (ZM-241385), ecto-5'-nucleotidase (α,β-methylene-adenosine diphosphate), BK(Ca) channels (paxilline), and K(ir) channels (BaCl(2)). Individually, these interventions led to 53-66% reductions in SNS-induced PADs. Combined applications of these blockers led to little or no further repression of SNS-induced PADs, suggesting interactions among A(2)Rs and K(+) channels. In the absence of SNS, BaCl(2) blockade of K(ir) channels produced 52-80% reductions in Ado and NS-1619 (BK(Ca) channel activator)-induced PADs. In contrast, paxilline blockade of BK(Ca) channels was without effect on dilations elicited by KCl (K(ir) channel activator) and Ado suffusions, indicating that Ado- and NS-1619-associated PADs involved K(ir) channels. In addition, targeted ablation of the superficial glia limitans was associated with a selective 60-80% loss of NS-1619 responses, suggesting that the BK(Ca) channel participation (and paxilline sensitivity) derived largely from channels within the glia limitans. Additionally, blockade of either PKA or adenylyl cyclase caused markedly attenuated pial arteriolar responses to SNS and, in the absence of SNS, responses to Ado, KCl, and NS-1619. These findings suggested a key, possibly permissive, role for A(2)R-linked cAMP generation and PKA-induced K(+) channel phosphorylation in somatosensory activation-evoked PAD.     

N-methyl-D-aspartate-induced vasodilation is mediated by endothelium-independent nitric oxide release in piglets

N-methyl-D-aspartate (NMDA) elicits pial arteriolar dilation that has been associated with neuronal nitric oxide (NO) production. However, endothelial factors or glial P-450 epoxygenase products may play a role. We tested whether NMDA-induced pial vasodilation 1) primarily involves NO diffusion from the parenchyma to the surface arterioles, 2) involves intact endothelial function, and 3) involves a miconazole-sensitive component. Arteriolar diameters were determined using closed cranial window-intravital microscopy in anesthetized piglets. NMDA (10-100 microM) elicited virtually identical dose-dependent dilations in paired arterioles (r = 0.94, n = 15). However, NMDA- but not bradykinin (BK)-induced dilations of arteriolar sections over large veins were reduced by 31 +/- 1% (means +/- SE, P < 0.05, n = 4) compared with adjacent sections on the cortical surface. Also, 100 microM NMDA increased cerebrospinal fluid levels of NO metabolites from 3.7 +/- 1.0 to 5.3 +/- 1.2 microM (P < 0.05, n = 6). Endothelial stunning by intracarotid injection of phorbol 12,13-dibutyrate did not affect NMDA-induced vasodilation but attenuated vascular responses to hypercapnia and BK by approximately 70% (n = 7). Finally, miconazole (n = 6, 20 microM) pretreatment and coapplication with NMDA did not alter vascular responses to NMDA. In conclusion, NMDA appears to dilate pial arterioles exclusively through release and diffusion of NO from neurons to the pial surface in piglets.     

Role of 20-HETE in the pial arteriolar constrictor response to decreased hematocrit after exchange transfusion of cell-free polymeric hemoglobin.

The cerebrovascular response to decreases in hematocrit and viscosity depends on accompanying changes in arterial O2 content. This study examines whether 1) the arteriolar dilation seen after exchange transfusion with a 5% albumin solution can be reduced by the K(ATP) channel antagonist glibenclamide (known to inhibit hypoxic dilation), and 2) the arteriolar constriction seen after exchange transfusion with a cell-free hemoglobin polymer to improve O2-carrying capacity can be blocked by inhibitors of the synthesis or vasoconstrictor actions of 20-HETE. In anesthetized rats, decreasing hematocrit by one-third with albumin exchange transfusion dilated pial arterioles (14 +/- 2%; SD), whereas superfusion of the surface of the brain with 10 muM glibenclamide blocked this response (-10 +/- 7%). Exchange transfusion with polymeric hemoglobin decreased the diameter of pial arterioles by 20 +/- 3% without altering arterial pressure. This constrictor response was attenuated by superfusing the surface of the brain with a 20-HETE antagonist, WIT-002 (10 microM; -5 +/- 1%), and was blocked by two chemically dissimilar selective inhibitors of the synthesis of 20-HETE, DDMS (50 microM; 0 +/- 4%) and HET-0016 (1 microM; +6 +/- 4%). The constrictor response to hemoglobin transfusion was not blocked by an inhibitor of nitric oxide (NO) synthase, and the inhibition of the constrictor response by DDMS was not altered by coadministration of the NO synthase inhibitor. We conclude 1) that activation of K(ATP) channels contributes to pial arteriolar dilation during anemia, whereas 2) constriction to polymeric hemoglobin transfusion at reduced hematocrit represents a regulatory response that limits increased O2 transport and that is mediated by increased formation of 20-HETE, rather than by NO scavenging.     

Adenosine A(2A) receptors mediate cardiovascular responses to hypoxia in fetal sheep

Nonselective adenosine (ADO) receptor antagonists block hypoxia-induced bradycardia and hypertension in fetal sheep. This study was designed to determine the ADO receptor subtype that is involved in these cardiovascular responses. In chronically catheterized fetal sheep (>0.8 term), fetal hypoxemia was induced by having the ewe breathe a hypoxic gas mixture (9% O(2)-3% CO(2)-88% N(2)) for 1 h. Intra-arterial infusion of ZM-241385, an antagonist highly selective for ADO A(2A) receptors, to eight fetuses during normoxia significantly increased mean arterial pressure (MAP) from 42.5 +/- 2.0 to 46.1 +/- 2.0 mmHg without altering heart rate (HR). Infusion of a selective antagonist of ADO A(1) receptors [1, 3-dipropyl-8-cyclopentylxanthine (DPCPX)] elevated MAP and HR only after the infusion was terminated, although administration of the vehicle for ZM-241385 or DPCPX had no effect on MAP or HR. Isocapnic hypoxia with infusion of DPCPX or the vehicle for DPCPX or ZM-241385 produced a transient fall in HR, a rise in MAP, and a decrease in plasma volume. In contrast, ADO A(2A) receptor blockade abolished the hypoxia-induced bradycardia and hypertension and blunted the decline in plasma volume. We conclude that fetal ADO A(2A) receptors: 1) modulate AP during normoxia, and 2) mediate cardiovascular responses during acute O(2) deficiency.     

Pial arteriole dilation during somatosensory stimulation is not mediated by an increase in CSF metabolites

Pial arterioles supplying the hindlimb somatosensory cortex dilate in response to contralateral sciatic nerve stimulation. The mechanism of this pial vasodilation is not well understood. One possibility is that vasoactive metabolites released during brain activation may diffuse to subarachnoid cerebrospinal fluid (CSF) to dilate pial vessels. To test this hypothesis, we implanted closed cranial windows in rats and measured pial arteriolar dilation to sciatic nerve stimulation during constant rate superfusion of the pial surface with artificial CSF. We reason that flushing the pial surface with CSF should quickly dissipate vasoactive substances and prevent these substances from dilating pial arterioles. CSF flow (1 and 1.5 ml/min) significantly reduced pial arteriole dilation induced by 5% CO2 inhalation, but the same flow rates did not affect dilator responses to sciatic nerve stimulation. We conclude that brain-to-CSF diffusion of vasoactive metabolites does not play a significant role in the dilation of pial arterioles during somatosensory activity.     

Carbon monoxide contributes to hypotension-induced cerebrovascular vasodilation in piglets

The gaseous compound carbon monoxide (CO) has been identified as an important endogenous biological messenger in brain and is a major component in regulation of cerebrovascular circulation in newborns. CO is produced endogenously by catabolism of heme to CO, free iron, and biliverdin during enzymatic degradation of heme by heme oxygenase (HO). The present study was designed to test the hypothesis that endogenously produced CO contributes to hypotension-induced vasodilation of cerebral arterioles. Experiments used anesthetized piglets with implanted, closed cranial windows. Topical application of the HO substrate heme-l-lysinate caused dilation of pial arterioles that was blocked by a metal porphyrin inhibitor of HO, chromium mesoporphyrin (CrMP). In normotensive piglets (arterial pressure 64 +/- 4 mmHg), CrMP did not cause vasoconstriction of pial arterioles but rather a transient dilation. Hypotension (50% of basal blood pressure) increased cerebral CO production and dilated pial arterioles from 66 +/- 2 to 92 +/- 7 microm. In hypotensive piglets, topical CrMP or intravenous tin protoporphyrin decreased cerebral CO production and produced pial arteriolar constriction to normotensive diameters. In additional experiments, because prostacyclin and nitric oxide (NO) are also key dilators that can contribute to cerebrovascular dilation, we held their levels constant. NO/prostacyclin clamp was accomplished with continuous, simultaneous application of indomethacin, N(omega)-nitro-l-arginine, and minimal dilatory concentrations of iloprost and sodium nitroprusside. With constant NO and prostacyclin, the transient dilator and prolonged constrictor responses to CrMP of normotensive and hypotensive piglets, respectively, were the same as when NO and prostaglandins were not held constant. These data suggest that endogenously produced CO contributes to cerebrovascular dilation in response to reduced perfusion pressure.     

Angiotensin II type 1 (AT1)-receptor blocker prevents impairment of endothelium-dependent cerebral vasodilation by acute cigarette smoking in rats

Our aim was to test for smoking-induced endothelial dysfunction in rat cerebral vessels, then to evaluate the effect of valsartan [angiotensin II type I (AT1)-receptor blocker] on that impairment. In pentobarbital-anesthetized, mechanically ventilated Sprague-Dawley rats, we used a cranial window preparation to measure changes in pial vessel diameters following topical applications of acetylcholine (Ach) (before and after smoking or intravenous nicotine infusion; n = 6 in each group), and adenosine (n = 6 for before and after smoking). Then, after intravenous valsartan pretreatment we reexamined the pial vasodilator response to topical Ach (before and after cigarette smoking). Under control conditions, cerebral arterioles were dilated by 6.9 +/- 4.2% and 13.6 +/- 4.8% by topical Ach (10(-6) M and 10(-5) M, respectively) and by 6.4 +/- 2.5% and 12.2 +/- 3.1% by topical adenosine (10(-5) M and 10(-4) M, respectively). One hour after a 1-min inhalation of mainstream smoke (1-mg nicotine cigarette), 10(-5) M Ach constricted cerebral arterioles (-4.4 +/- 4.1%), while 10(-4) M adenosine dilated them by 13.4 +/- 3.4%. One hour after a 1-min nicotine infusion (0.05 mg), 10(-5) M Ach dilated cerebral arterioles by 9.9 +/- 2.4%. Thus, vasodilator response to topical Ach was impaired after smoking, whereas that to adenosine was unaffected. However, the vasodilator response to Ach was unaffected by intravenous nicotine. Valsartan prevented smoking from impairing Ach-induced vasodilation. In conclusion, acute single-cigarette smoking causes a dysfunction of endothelium-dependent, but not endothelium-independent, vasodilation of rat cerebral vessels in vivo, and the effect was not mimicked by intravenous nicotine. AT1-receptor blockade prevented the above smoking-induced impairment of endothelium-dependent vasodilation.     

Brimonidine evokes heterogeneous vasomotor response of retinal arterioles: diminished nitric oxide-mediated vasodilation when size goes small

Brimonidine, an alpha2-adrenergic receptor (AR) agonist, has been employed in the treatment of glaucoma due to its beneficial effects on intraocular pressure reduction and neuroprotection. In addition, some studies have implicated that brimonidine might influence ocular blood flow; however, its effect on the retinal microcirculation has not been documented. Herein, we examined the vasomotor action of brimonidine on different branching orders of retinal arterioles in vitro and determined the contribution of the alpha2-AR subtype and the role of endothelium-derived nitric oxide (NO) in this vasomotor response. First- and second-order retinal arterioles of pigs were isolated, cannulated, and pressurized for functional studies. Videomicroscopic techniques were employed to record diameter changes in response to brimonidine. RT-PCR was performed for detection of alpha-AR and endothelial NO synthase (eNOS) mRNA in retinal arterioles. All first-order arterioles (82 +/- 2 microm ID) dilated dose dependently to brimonidine (0.1 nM to 10 microM) with 10% dilation at the highest concentration. Second-order arterioles (50 +/- 1 microm ID) responded heterogeneously with either dilation or constriction. The incidence and magnitude of vasoconstriction were increased with increasing brimonidine concentration. Administration of the NO synthase inhibitor NG-nitro-L-arginine methyl ester abolished the brimonidine-induced vasodilation in first- and second-order arterioles. Regardless of vessel size, vasomotor responses (i.e., vasodilation and vasoconstriction) of retinal arterioles were sensitive to the alpha2-AR antagonist rauwolscine. Consistent with the functional data, alpha2A-AR and eNOS mRNAs were detected in retinal arterioles. Collectively, our data demonstrate that brimonidine at clinical doses evokes a consistent NO-dependent vasodilation in first-order retinal arterioles but a heterogeneous response in second-order arterioles. These vasomotor responses are mediated by the activation of alpha2-AR. It appears that brimonidine, depending on the concentration and vessel size, may alter local retinal blood flow.     

Abolition of arteriolar dilation but not constriction to histamine in cremaster muscle of eNOS-/- mice

Histamine increases the permeability of capillaries and venules but little is known of its precapillary actions on the control of tissue perfusion. Using gene ablation and pharmacological interventions, we tested whether histamine could increase muscle blood flow through stimulating nitric oxide (NO) release from microvascular endothelium. Vasomotor responses to topical histamine were investigated in second-order arterioles in the superfused cremaster muscle of anesthetized C57BL6 mice and null platelet endothelial cell adhesion molecule-1 (PECAM-1-/-) and null endothelial NO synthase (eNOS-/-) mice aged 8-12 wk. Neither resting (17 +/- 1 microm) nor maximum diameters (36 +/- 2 microm) were different between groups, nor was the constrictor response (approximately 5 +/- 1 microm) to elevating superfusate oxygen from 0 to 21%. For arterioles of C57BL6 and PECAM-1-/- mice, cumulative addition of histamine to the superfusate produced vasodilation (1 nM-1 microM; peak response, 9 +/- 1 microm) and then vasoconstriction (10-100 microM; peak response, 12 +/- 2 microm). In eNOS-/- mice, histamine produced only vasoconstriction. In C57BL6 and PECAM-1-/- mice, vasodilation was abolished with Nomega-nitro-l-arginine (30 microM); in all mice, vasoconstriction was abolished with nifedipine (1 microM). Vasomotor responses were eliminated with pyrilamine (1 microM; H1 receptor antagonist) yet remained intact with cimetidine (1 microM; H2 receptor antagonist). These findings illustrate that the biphasic vasomotor response of mouse cremaster arterioles to histamine is mediated through H1 receptors on endothelium (NO-dependent vasodilation) as well as smooth muscle (Ca2+ entry and constriction). Thus histamine can increase as well as decrease muscle blood flow, according to local concentration. However, when NO production is compromised, only vasoconstriction and flow reduction occur.     

Hydrogen peroxide acts as an EDHF in the piglet pial vasculature in response to bradykinin

We investigated the mechanism of EDHF-mediated dilation to bradykinin (BK) in piglet pial arteries. Topically applied BK (3 micromol/l) induced vasodilation (62 +/- 12%) after the administration of N(omega)-nitro-L-arginine methyl ester (L-NAME) and indomethacin, which was inhibited by endothelial impairment or by the BK(2) receptor antagonist HOE-140 (0.3 micromol/l). Western blotting showed the presence of BK(2) receptors in brain cortex and pial vascular tissue samples. The cytochrome P-450 antagonist miconazole (20 micromol/l) and the lipoxygenase inhibitors baicalein (10 micromol/l) and cinnamyl-3,4-dyhydroxy-alpha-cyanocinnamate (1 micromol/l) failed to reduce the BK-induced dilation. However, the H(2)O(2) scavenger catalase (400 U/ml) abolished the response (from 54 +/- 11 to 0 +/- 2 microm; P < 0.01). The ATP-dependent K(+) (K(ATP)) channel inhibitor glibenclamide (10 micromol/l) had a similar effect as well (from 54 +/- 11 to 16 +/- 5 microm; P < 0.05). Coapplication of the Ca(2+)-dependent K(+) channel inhibitors charybdotoxin (0.1 micromol/l) and apamin (0.5 micromol/l) failed to reduce the response. We conclude that H(2)O(2) mediates the non-nitric oxide-, non-prostanoid-dependent vasorelaxation to BK in the piglet pial vasculature. The response is mediated via BK(2) receptors and the opening of K(ATP) channels.     

Role of cGMP in carbon monoxide-induced cerebral vasodilation in piglets

The hypothesis was addressed that CO-induced cerebral vasodilation requires a permissive cGMP signal that can be produced by nitric oxide (NO). Anesthetized piglets were implanted with cranial windows for measurement of pial arteriolar responses to stimuli. Pial arterioles dilated in response to isoproterenol (Iso), sodium nitroprusside (SNP), and CO or the CO-releasing molecule Mn2(CO)10 [dimanganese decacarbonyl (DMDC)]. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylyl cyclase inhibitor, decreased cerebrospinal fluid (CSF) cGMP and selectively inhibited dilations to SNP and DMDC without affecting the dilation to Iso. However, DMDC did not cause an increase in cortical periarachnoid CSF cGMP concentration. cGMP clamp with a threshold dilator level of 8-bromo-cGMP (10(-4) M) and ODQ restored the dilation to DMDC that had been blocked by ODQ alone. Under these conditions, cGMP was present but could not increase. Inhibition of the pial arteriolar dilation to glutamate by N-nitro-l-arginine, which blocks NO synthase, was similar to that by heme oxygenase inhibitors, which block endogenous CO production. The dilation to glutamate, similar to dilation to DMDC, was partially restored by 8-bromo-cGMP and completely restored by SNP (5 x 10(-7) M). These data suggest that the permissive role of NO in CO- and glutamate-induced vasodilation involves maintaining the minimum necessary cellular level of cGMP to allow CO to cause dilation independently of increasing cGMP.