Tone Of Parenchymal Arterioles Research Articles

Decreased parenchymal arteriolar tone uncouples vessel-to-neuronal communication in a mouse model of vascular cognitive impairment

Chronic hypoperfusion is a key contributor to cognitive decline and neurodegenerative conditions, but the cellular mechanisms remain ill-defined. Using a multidisciplinary approach, we sought to elucidate chronic hypoperfusion-evoked functional changes at the neurovascular unit. We used bilateral common carotid artery stenosis (BCAS), a well-established model of vascular cognitive impairment, combined with an ex vivo preparation that allows pressurization of parenchymal arterioles in a brain slice. Our results demonstrate that mild (~ 30%), chronic hypoperfusion significantly altered the functional integrity of the cortical neurovascular unit. Although pial cerebral perfusion recovered over time, parenchymal arterioles progressively lost tone, exhibiting significant reductions by day 28 post-surgery. We provide supportive evidence for reduced adenosine 1 receptor-mediated vasoconstriction as a potential mechanism in the adaptive response underlying the reduced baseline tone in parenchymal arterioles. In addition, we show that in response to the neuromodulator adenosine, the action potential frequency of cortical pyramidal neurons was significantly reduced in all groups. However, a significant decrease in adenosine-induced hyperpolarization was observed in BCAS 14 days. At the microvascular level, constriction-induced inhibition of pyramidal neurons was significantly compromised in BCAS mice. Collectively, these results suggest that BCAS uncouples vessel-to-neuron communication—vasculo-neuronal coupling—a potential early event in cognitive decline.

Differential expression of angiotensin II type 1 receptor subtypes within the cerebral microvasculature.

Arteries and arterioles constrict in response to intraluminal pressure to generate myogenic tone, but the molecular nature of the vascular force-sensing mechanism is not fully characterized. Here, we investigated the role of angiotensin II type 1 receptors (AT1Rs) on vascular smooth muscle cells in the development of myogenic tone in cerebral parenchymal arterioles from mice. We found that pretreatment with the AT1R blocker losartan inhibited the development of myogenic tone in these vessels but did not alter the luminal diameter of arterioles with preestablished tone. Rodents express two AT1R isotypes: AT1Ra and AT1Rb. We previously demonstrated that AT1Rb is expressed at much higher levels compared with AT1Ra in cerebral pial arteries and is required for myogenic contractility in these vessels, whereas AT1Ra is unnecessary for this function. Here, we found that AT1Ra and AT1Rb are expressed at similar levels in parenchymal arterioles and that genetic knockout of AT1Ra blunted the ability of these vessels to generate myogenic tone. We also found that AT1Rb and total AT1R expression levels are much lower in parenchymal arterioles compared with pial arteries and that parenchymal arterioles are less sensitive to the vasoconstrictive effects of the endogenous AT1R ligand angiotensin II (ANG II). We conclude that 1) AT1Rs are critical for the initiation, but not the maintenance, of myogenic tone in parenchymal arterioles, and 2) lower levels of AT1Rb and total AT1R in parenchymal arterioles compared with pial arteries result in differences in myogenic and ANG II-induced vasoconstriction between these vascular segments.NEW & NOTEWORTHY Myogenic tone is critical for appropriate regulation of cerebral blood flow, but the mechanisms used by vascular smooth muscle cells to detect changes in intraluminal pressure are not fully characterized. Here, we demonstrate angiotensin II receptor type 1 (AT1R) is indispensable to initiation, but not maintenance, of myogenic tone in cerebral parenchymal arterioles. Furthermore, we demonstrate differences in AT1R expression levels lead to critical differences in contractile regulation between parenchymal arterioles and cerebral pial arteries.

Transient receptor potential vanilloid-4 channels are involved in diminished myogenic tone in brain parenchymal arterioles in response to chronic hypoperfusion in mice.

Adaptive responses of brain parenchymal arterioles (PAs), a target for cerebral small vessel disease, to chronic cerebral hypoperfusion are largely unknown. Previous evidence suggested that transient receptor potential vanilloid 4 channels may be involved in the regulation of cerebrovascular tone. Therefore, we investigated the role of TRPV4 in adaptations of PAs in a mouse model of chronic hypoperfusion. TRPV4 knockout (-/- ) and wild-type (WT) mice were subjected to unilateral common carotid artery occlusion (UCCAo) for 28days. Function and structure of PAs ipsilateral to UCCAo were studied isolated and pressurized in an arteriograph. Basal tone of PAs was similar between WT and TRPV4-/- mice (22±3 vs 23±5%). After UCCAo, active inner diameters of PAs from WT mice were larger than control (41±2 vs 26±5μm, P<0.05) that was due to decreased tone (8±2 vs 23±5%, P<0.05), increased passive inner diameters (46±3 vs 34±2μm, P<0.05), and decreased wall-to-lumen ratio (0.104± 0.01 vs 0.137±0.01, P<0.05). However, UCCAo did not affect vasodilation to a small- and intermediate-conductance calcium-activated potassium channel agonist NS309, the nitric oxide (NO) donor sodium nitroprusside, or constriction to a NO synthase inhibitor L-NNA. Wall thickness and distensibility in PAs from WT mice were unaffected. In TRPV4-/- mice, UCCAo had no effect on active inner diameters or tone and only increased passive inner diameters (53±2 vs 43±3μm, P<0.05). Adaptive response of PAs to chronic cerebral hypoperfusion includes myogenic tone reduction and outward remodelling. TRPV4 channels were involved in tone reduction but not outward remodelling in response to UCCAo.

Effect of hypertension and peroxynitrite decomposition with FeTMPyP on CBF and stroke outcome

We investigated the effect of peroxynitrite decomposition catalyst FeTMPyP treatment on perfusion deficit, vascular function and stroke outcome in Wistar (n = 26) and spontaneously hypertensive rats stroke-prone (SHRSP; n = 26) that underwent tMCAO for 2 h or Sham operation. Peri-infarct CBF was measured by hydrogen clearance in the absence or presence of FeTMPyP (10 mg/kg, i.v.) or vehicle 10 min before reperfusion. Myogenic tone of parenchymal arterioles (PAs) was measured as an indication of small vessel resistance (SVR). Baseline CBF was similar between Wistar and SHRSP (114 ± 12 vs. 132 ± 9 mL/100 g/min); however, MCAO caused greater perfusion deficit in SHRSP (24 ± 6 vs. 7 ± 1 mL/100 g/min; p < 0.05) and increased infarct volume by TTC (12 ± 6 vs. 32 ± 2%; p < 0.05). Reperfusion CBF was decreased from baseline in both SHRSP and Wistar (54 ± 16 and 46 ± 19 mL/100 g/min; p < 0.05), suggesting increased infarction in SHRSP was related to greater perfusion deficit. PAs from SHRSP had increased tone vs. Wistar that was enhanced after tMCAO. FeTMPyP treatment did not affect CBF during ischemia or reperfusion, or tone of PAs, but decreased the incidence of hemorrhage in SHRSP by 50%. Thus, increased tone in PAs from SHRSP could increase perfusion deficit during MCAO that was not alleviated by FeTMPyP.

Effects of Early Post-Ischemic Reperfusion and tPA on Cerebrovascular Function and Nitrosative Stress in Female Rats.

Stroke is a major health issue in women. Our previous studies in male rats showed decreased myogenic tone in middle cerebral arteries (MCAs) after ischemia and reperfusion (I/R), while tone in parenchymal arterioles (PAs) was increased. This vascular response may aggravate stroke damage in males by limiting reperfusion; however, the effect in females is not known. The current study investigated the effect of I/R and tissue plasminogen activator (tPA) on myogenic tone and reactivity of MCAs and PAs in female rats. Nitrosative stress by peroxynitrite and recruitment of inflammatory neutrophils to the microvasculature were also studied. Female rats were subjected to 2-h MCA filament occlusion (n = 16) or sham surgery (n = 17) and given tPA (1mg/kg, i.v) or vehicle followed by 30-min reperfusion. Myogenic tone and reactivity were measured in isolated and pressurized MCAs and PAs from the same animals. Cerebrovascular F-actin, 3-nitrotyrosine (3-NT, peroxynitrite marker), and intravascular neutrophils were quantified. Myogenic tone and constriction to the nitric oxide synthase inhibitor Nω-nitro-L-arginine were decreased in MCAs but unchanged in PAs after I/R with no effect of tPA. F-actin and 3-NT expression were unaffected by I/R or tPA. Our study showed that MCAs from females, similar to what has been seen in males, are dilated after I/R and have decreased myogenic tone while tone in PAs was unchanged. Increased small vessel resistance may contribute to decreased reperfusion and worse outcome after stroke.

Treatment of Acute Focal Ischemic Stroke with Peroxynitrite Decomposition Catalyst does not Improve Reperfusion, Infarct or Hemorrhage in Chronic Hypertensive Rats

Amajority of acute ischemic stroke patients has a history of chronic hypertension, which is associated with worse stroke outcome. Previously we found that ischemia and reperfusion enhanced myogenic tone in brain parenchymalarterioles (PAs) from normotensive rats, however, the impact of chronic hypertension on these vessels after acute ischemic stroke is unknown. We hypothesized that chronic hypertension further increases myogenic tone of PAs after ischemia and reperfusion that worsens early post‐ischemic reperfusion, leading to worse stroke outcome. Moreover, we previously found that the peroxynitrite decomposition catalyst FeTMPyP decreased myogenic tone of PAs after ischemia and reperfusion. We therefore further hypothesized that FeTMPyP treatment would improve reperfusion and hence stroke outcome. Male spontaneously hypertensive rats stroke‐prone (SHRSP, 18‐weeks) and age‐matched normotensive Wistar control(n=6–8/group) were subjected to transient middle cerebral artery occlusion or Sham operation. PAs, isolated after 2 hr of ischemia and 30 min of reperfusion, were pressurized in an arteriograph to study myogenic activity. In another set of animals, FeTMPyP (10 mg/kg, i.p.) or vehicle was given 10 min before reperfusion in Wistar or SHRSP. Early post‐ischemic reperfusion blood flow inbrain parenchyma within the peri‐infarct region was measured in anesthetized animals by hydrogen clearance. Brain infarct size and swelling after 2 hr of ischemia and 2 hr of reperfusion were determined by 2,3,5‐triphenyltetrazoliumchloride (TTC) staining. PAs from Sham SHRSP had increased myogenic tone compared to Sham Wistar (52 ± 2 vs. 43 ± 2 %; p&lt;0.05). Ischemia and reperfusion increased myogenic tone in PAs from Wistar but did not further increase tone in SHRSP, leading to similar tone between groups (59 ± 2 vs. 52 ± 2 %; p&gt;0.05). Brainparenchymal blood flow at 30 min reperfusion was significantly lower than base line in Wistar (54 ± 14 vs. 114 ± 13 ml/100 g tissue/min; p&lt;0.05) and SHRSP (43 ±15 vs. 132 ± 9 ml/100 g tissue/min; p&lt;0.05). However, reperfusion blood flow was similar between SHRSP and Wistar (43 ± 15 vs. 54 ± 14 ml/100 g tissue/min in Wistar). FeTMPyP did not alter reperfusion blood flow in Wistar (42 ± 9 vs. 54± 14 ml/100 g tissue/min in vehicle; p&gt;0.05) and SHRSP (30 min: 28 ± 5 vs. 43± 15 ml/100 g tissue/min in vehicle; p&gt;0.05). Compared to Wistar, SHRSP had increased brain infarct volume (31 ± 6 vs. 19 ± 6 % in Wistar; p&lt;0.05) and swelling (6± 2 vs. 12 ± 2 % in Wistar; p&lt;0.05). Hemorrhage within the infarct area was only found in SHRSP (~50 % of animals). FeTMPyP did not affect infarct volume, swelling, and incidence of hemorrhage in Wistar and SHRSP. Linear regression showed that early post‐ischemic reperfusion blood flow was related to infarctsize, with negative Pearson correlation coefficient in both Wistar (−0.472) and SHRSP (−0.694). In conclusion, worse stroke outcome in chronic hypertension does not appear to be related to further increase in myogenic tone of PA and worse reperfusion. Treatment with peroxynitrite decomposition catalyst does not improve or worsen reperfusion and stroke outcome.Support or Funding InformationThis study was supported by NIH NINDS Grant R01 NS‐093289 and NIH NHLBI Grant P01 HL‐095488.

Abstract 171: The Effect of Ischemia and Reperfusion and Tissue Plasminogen Activator on Cerebrovascular Function, Oxidative Stress and Polymorphonuclear Neutrophils in Ovariectomized Female Rats

Introduction: Stroke is a growing health problem in women. We previously showed that ischemia/reperfusion (I/R) in male rats reduced tone in middle cerebral arteries (MCA) but enhanced tone in parenchymal arterioles (PA). Tissue plasminogen activator (tPA) may contribute to vascular dysfunction, oxidative stress and inflammation. However, vascular dysfunction and the effect of tPA have not been studied in females. Hypothesis: I/R in ovariectomized (OVX) female rats impairs MCA and PA function differentially, increases vascular oxidative stress and intravascular polymorphonuclear neutrophils (PMN). tPA has an additive effect on the vascular dysfunction after I/R. Methods: OVX female rats underwent 2 h MCA filament occlusion (n=16) or sham surgery (n=17) with 30 min reperfusion. tPA (1 mg/kg, i.v.) or vehicle (Vh) was given 10 min prior to reperfusion. Myogenic tone and reactivity to nitric oxide synthase (L-NNA, 10-4M) and Rho kinase (Y27632, 10-8 to 10-5M) inhibition were measured in pressurized MCA and PA. MCA were stained for F-actin and 3-nitrotyrosine (NT), a marker of oxidative stress. Brain intravascular PMN were identified with myeloperoxidase and collagen IV using immunohistochemistry, and counted blinded to group. Results: In MCA, I/R decreased tone at 75 mmHg (Sham Vh: 26±3%, Sham tPA: 27±3% vs. MCAO Vh: 21±2%, MCAO tPA: 21±3%, P&lt;0.05) and reduced constriction to L-NNA (Sham Vh: 35±3%, Sham tPA: 34±2% vs. MCAO Vh: 20±3%, MCAO tPA: 25±3%, P&lt;0.01) with no effect on dilation to Y27632. In PA, myogenic tone (at 40 mmHg) and reactivity to L-NNA and Y27632 was not affected by I/R. PA were less sensitive to Y27632 than MCA after I/R with tPA (EC50: 2.0±0.5 vs. 0.8±0.1 μM, P&lt;0.05). F-actin and NT in the MCA were not affected by I/R or tPA. The number of PMN was small (&lt;0.8±0.3/mm2) in all groups but there was a trend to increased ipsilateral intravascular PMN after I/R with tPA. Conclusions: The differential effect of I/R on MCA vs. PA in OVX females on tone suggest parenchymal blood flow may be restricted during reperfusion that could increase perfusion deficits. tPA had no effect on tone in MCA and PA but might enhance intravascular PMN, implicated in no-reflow after I/R. Therapies to alleviate parenchymal vasoconstriction may improve stroke outcome in females.

Increased pressure-induced tone in rat parenchymal arterioles vs. middle cerebral arteries: role of ion channels and calcium sensitivity.

Brain parenchymal arterioles (PAs) are high-resistance vessels that branch off pial arteries and perfuse the brain parenchyma. PAs are the target of cerebral small vessel disease and have been shown to have greater pressure-induced tone at lower pressures than pial arteries. We investigated mechanisms by which brain PAs have increased myogenic tone compared with middle cerebral arteries (MCAs), focusing on differences in vascular smooth muscle (VSM) calcium and ion channel function. The amount of myogenic tone and VSM calcium was measured using Fura 2 in isolated and pressurized PAs and MCAs. Increases in intraluminal pressure caused larger increases in tone and cytosolic calcium in PAs compared with MCAs. At 50 mmHg, myogenic tone was 37 ± 5% for PAs vs. 6.5 ± 4% for MCAs (P < 0.01), and VSM calcium was 200 ± 20 nmol/l in PAs vs. 104 ± 15 nmol/l in MCAs (P < 0.01). In vessels permeabilized with Staphylococcus aureus α-toxin, PAs were not more sensitive to calcium, suggesting calcium sensitization was not at the level of the contractile apparatus. PAs were 30-fold more sensitive to the voltage-dependent calcium channel (VDCC) inhibitor nifedipine than MCAs (EC50 for PAs was 3.5 ± 0.4 vs. 82.1 ± 2.1 nmol/l for MCAs;P < 0.01); however, electrophysiological properties of the VDCC were not different in VSM. PAs had little to no response to the calcium-activated potassium channel inhibitor iberiotoxin, whereas MCAs constricted ∼15%. Thus increased myogenic tone in PAs appears related to differences in ion channel activity that promotes VSM membrane depolarization but not to a direct sensitization of the contractile apparatus to calcium.

Contribution of Rho kinase to TRPM4‐mediated myogenic tone in cerebral parenchymal arterioles (841.5)

Cerebral parenchymal arterioles (PAs) play a critical role in assuring appropriate blood flow and perfusion pressure within the brain. PAs are unique in contrast to cerebral pial arteries, as defined by their critical roles in neurovascular coupling and distinct sensitivities to mechanical and chemical stimulants. Elevation in intraluminal pressure, a physiologic vasomotor stimulant, causes smooth muscle membrane depolarization and contraction (myogenic tone) in cerebral arteries. However, the mechanisms by which this myogenic tone is regulated in the cerebral microcirculation remain unclear. Prior research in our laboratory indicates that myogenic tone of PAs is mediated primarily through stimulation of mechano‐sensitive P2Y purinergic receptors. The objective of the present study was to determine the roles of TRPM4 channels and Rho kinase signaling in PA myogenic tone. We show that suppression of TRPM4 channel expression in vivo using antisense oligonucleotides reduced (by ~40%) both myogenic and P2Y‐receptor agonist‐induced tone in PAs. Further, we found that the Rho kinase inhibitor H1152 reversibly and nearly completely inhibited myogenic tone in these brain arterioles (IC50:0.2 µM). H1152 (1 µM) also attenuated P2Y4 and P2Y6 receptor‐mediated vasoconstriction by 85% and 87%, respectively. Finally, H1152 (1 μM) reduced constitutive and UTPγS (0.5 μM)‐activated TRPM4 currents in parenchymal myocytes by 61% and 75%, respectively. These results indicate an important role for Rho kinase in regulation of TRPM4 channel activity and myogenic tone in the cerebral microcirculation.Grant Funding Source: Supported by NIH HL095488

Mechanisms of Enhanced Basal Tone of Brain Parenchymal Arterioles During Early Postischemic Reperfusion: Role of ET-1-Induced Peroxynitrite Generation

The contributions of vasoconstrictors (endothelin-1 (ET-1), peroxynitrite) and endothelium-dependent vasodilatory mechanisms to basal tone were investigated in parenchymal arterioles (PAs) after early postischemic reperfusion. Transient middle cerebral artery occlusion (tMCAO) was induced for 2 hours with 30 minutes reperfusion in male Wistar rats and compared with ischemia alone (permanent MCAO (pMCAO); 2.5 hours) or sham controls. Changes in lumen diameter of isolated and pressurized PAs were compared. Quantitative PCR was used to measure endothelin type B (ETB) receptors. Constriction to intravascular pressure ('basal tone') was not affected by tMCAO or pMCAO. However, constriction to inhibitors of endothelial cell, small- (SK) and intermediate- (IK) conductance, Ca(2+)-sensitive K(+) channels (apamin and TRAM-34, respectively) were significantly enhanced in PAs from tMCAO compared with pMCAO or sham. Addition of the ETB agonist sarafotoxin caused constriction in PAs from tMCAO but not from sham animals (21 ± 4% versus 3 ± 3% at 1 nmol/L; P<0.01) that was inhibited by the peroxynitrite scavenger FeTMPyP (5,10,15,20-tetrakis (N-methyl-4'-pyridyl) porphinato iron (III) chloride) (100 μmol/L). Expression of ETB receptors was not found on PA smooth muscle, suggesting that constriction to sarafotoxin after tMCAO was due to peroxynitrite and not ETB receptor expression. The maintenance of basal tone in PAs after tMCAO may restrict flow to the ischemic region and contribute to infarct expansion.

The Same but Different

Individual segments along the cerebrovascular tree serve unique functions in the overall coordination of cerebral blood flow. Consequently, after pathologic conditions, we must understand how each segment of the cerebrovascular tree is affected in order to fully comprehend the net effect on cerebral blood flow. In this issue, Cipolla et al.1 studied parenchymal arterioles (PAs), a final control point before capillaries in the cerebral cortex, early into the reperfusion phase after an ischemic event. Reperfusion is the beginning of a pathologic cascade that amplifies tissue damage. The PAs, which penetrated into the cortex from a branch of the middle cerebral artery, were isolated and pressurized to study the development of spontaneous tone, an intrinsic property of arteries and arterioles where the vessels constrict to pressures across the vessel wall.2 PAs that were isolated 30 minutes into reperfusion developed the same amount of tone as PAs from naive rats over intraluminal pressures ranging from 40 to 80 mm Hg. Thus, without further investigations, PAs after ischemia/reperfusion (I/R) might be considered ‘the same' as those without insult. However, after further probing, the investigator discovered that after I/R, PAs maintained ‘the same' tone as naive PAs but did so using ‘different' mechanisms for achieving this tone. First, there was an enhanced dilator response through enhanced endothelial-dependent hyperpolarization (EDH), a process where hyperpolarizations of the endothelium by small and intermediate conductance calcium-activated potassium channels are passed to the vascular smooth muscle (VSM) through gap junctions.3 Hyperpolarizations of the VSM, in turn, close voltage-operated calcium channels to dilate the arteriole. With this enhanced dilator process, another mechanism(s) must be present to constrict PAs in an equal and opposite manner to EDH. Although the authors cannot definitively state what underlies this constrictors process, they did show constrictor responses with activation of the endothelin receptor type B (ETBR) only after I/R. As message for ETBR was not found in VSM of the PAs, the ETBR-related constriction must have involved the endothelium. ETBR on endothelium are known to activate endothelial nitric oxide (NO) synthase.4, 5, 6 The investigators speculated that reactive oxygen species associated with I/R reacted with newly synthesized NO after ETBR stimulation to produce peroxynitrite. Peroxynitrite was demonstrated in this study to constrict PAs through an unknown mechanism. Although the investigators provided interesting and provocative circumstantial evidence for an ETBR constrictor response to offset the EDH dilatory effect, more studies will be required for a definitive conclusion. While, there are many questions remaining as to what is controlling the tone of PAs after I/R, the fact remains that the tone is the ‘same' as naive PAs but the mechanisms regulating this tone are ‘different'.

Purinergic Receptors Regulate Myogenic Tone in Cerebral Parenchymal Arterioles

Myogenic tone is a fundamental aspect of vascular behavior in resistance arteries. This contractile response to changes in intravascular pressure is critically involved in blood flow autoregulation in tissues such as the brain, kidneys, and heart. Myogenic tone also helps regulate precapillary pressure and provides a level of background tone upon which vasodilator stimuli act to increase tissue perfusion when appropriate. Despite the importance of these processes in the brain, little is known about the mechanisms involved in control of myogenic tone in the cerebral microcirculation. Here, we report that pharmacological inhibition of P2Y4 and P2Y6 pyrimidine receptors nearly abolished myogenic tone in cerebral parenchymal arterioles (PAs). Molecular suppression of either P2Y4 or P2Y6 receptors using antisense oligodeoxynucleotides reduced myogenic tone by 44%±8% and 45%±7%, respectively. These results indicate that both receptor isoforms are activated by increased intravascular pressure, which enhances the activity of voltage-dependent calcium channels and increases myogenic tone in PAs. Enhancement or inhibition of ectonucleotidase activity had no effect on parenchymal arteriolar myogenic tone, indicating that this response is not mediated by local release of nucleotides, but rather may involve direct mechanical activation of P2Y receptors in the smooth muscle cells.

SK Ca and IK Ca Channels, Myogenic Tone, and Vasodilator Responses in Middle Cerebral Arteries and Parenchymal Arterioles

The role of SK(Ca) and IK(Ca) channels in myogenic tone and endothelium-derived hyperpolarizing factor (EDHF) responsiveness was investigated under control conditions and after ischemia and reperfusion in parenchymal arterioles (PA) versus middle cerebral arteries (MCA). MCA and PA were dissected from male Wistar rats that were ischemic for 1 hour with 24 hours of reperfusion (n=12) or sham controls (n=12). Basal tone and reactivity to apamin (300 nmol/L), TRAM-34 (1.0 micromol/L), and nitro-L-arginine (0.1 mmol/L) were compared in PA and MCA pressurized to 40 mm Hg and 75 mm Hg, respectively. SK(Ca) and IK(Ca) channel mRNA expression was measured using real-time PCR. PA developed greater basal tone than MCA (42+/-4% versus 19+/-3%; P<0.01). Addition of apamin and TRAM-34 increased tone of PA by 25+/-3% and 16+/-2%, respectively, whereas MCA had no response to either inhibitor. After ischemia and reperfusion, the response to nitric oxide synthase inhibition (NOS) was diminished in PA, whereas EDHF responsiveness was preserved. In addition, stimulated EDHF dilation was partially reversed by apamin and completely reversed by TRAM-34 in both control and ischemic PA. SK(Ca) and IK(Ca) channel mRNA expression was similar in PA and MCA and not altered by ischemia and reperfusion. However, IK(Ca) channel mRNA expression was 4- to 5-fold greater than SK(Ca) channels. It appears that SK(Ca) and IK(Ca) channel activity diminishes basal tone of PA, but not MCA. The preservation of EDHF responsiveness of PA after ischemia and reperfusion suggests an important role for this vasodilator under conditions when NOS is inhibited.