Cerebrovascular response to decreased hematocrit: effect of cell-free hemoglobin,plasma viscosity,and CO2

The effect of transfusing a nonextravasating, zero-link polymer of cell-free hemoglobin on pial arteriolar diameter, cerebral blood flow (CBF), and O2 transport (CBF x arterial O2 content) was compared with that of transfusing an albumin solution at equivalent reductions in hematocrit (approximately 19%) in anesthetized cats. The influence of viscosity was assessed by coinfusion of a high-viscosity solution of polyvinylpyrrolidone (PVP), which increased plasma viscosity two- to threefold. Exchange transfusion of a 5% albumin solution resulted in pial arteriolar dilation, increased CBF, and unchanged O2 transport, whereas there were no significant changes over time in a control group. Exchange transfusion of a 12% polymeric hemoglobin solution resulted in pial arteriolar constriction and unchanged CBF and O2 transport. Coinfusion of PVP with albumin produced pial arteriolar dilation that was similar to that obtained with transfusion of albumin alone. In contrast, coinfusion of PVP with hemoglobin converted the constrictor response to a dilator response that prevented a decrease in CBF. Pial arteriolar dilation to hypercapnia was unimpaired in groups transfused with albumin or hemoglobin alone but was attenuated in the largest vessels in albumin and hemoglobin groups coinfused with PVP. Unexpectedly, hypocapnic vasoconstriction was blunted in all groups after transfusion of albumin or hemoglobin alone or with PVP. We conclude that 1) the increase in arteriolar diameter after albumin transfusion represents a compensatory response that prevents decreased O2 transport at reduced O2-carrying capacity, 2) the decrease in diameter associated with near-normal O2-carrying capacity after cell-free polymeric hemoglobin transfusion represents a compensatory mechanism that prevents increased O2 transport at reduced blood viscosity, 3) pial arterioles are capable of dilating to an increase in plasma viscosity when hemoglobin is present in the plasma, 4) decreasing hematocrit does not impair pial arteriolar dilation to hypercapnia unless plasma viscosity is increased, and 5) pial arteriolar constriction to hypocapnia is impaired at reduced hematocrit independently of O2-carrying capacity.     

The CYP450 hydroxylase pathway contributes to P2X receptor-mediated afferent arteriolar vasoconstriction

This study was conducted to test the hypothesis that the cytochrome P-450 (CYP450) metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to the afferent arteriolar response to P2 receptor activation. Afferent arteriolar responses to ATP, the P2X agonist, alpha,beta-methylene ATP and the P2Y agonist UTP were determined before and after treatment with the selective CYP450 hydroxylase inhibitor, N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) or the 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE). Stimulation with 1.0 and 10 microM ATP elicited an initial preglomerular vasoconstriction of 12 +/- 1% and 45 +/- 4% and a sustained vasoconstriction of 11 +/- 1% and 11 +/- 2%, respectively. DDMS or 20-HEDE significantly attenuated the sustained afferent arteriolar constrictor response to ATP. alpha,beta-Methylene ATP (1 microM) induced a rapid initial afferent vasoconstriction of 64 +/- 3%, which partially recovered to a stable diameter 10 +/- 1% smaller than control. Both DDMS and 20-HEDE significantly attenuated the initial vasoconstriction and abolished the sustained vasoconstrictor response to alpha,beta-methylene ATP. UTP decreased afferent diameter by 50 +/- 5% and 20-HEDE did not change this response. In addition, the ATP-induced increase in the intracellular Ca2+ concentration in preglomerular microvascular smooth muscle cells was significantly attenuated by 20-HEDE. Taken together, these results are consistent with the hypothesis that the CYP450 metabolite 20-HETE participates in the afferent arteriolar response to activation of P2X receptors.     

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.     

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 heme oxygenase-2 in pial arteriolar response to acetylcholine in mice with and without transfusion of cell-free hemoglobin polymers

Carbon monoxide derived from heme oxygenase (HO) may participate in cerebrovascular regulation under specific circumstances. Previous work has shown that HO contributes to feline pial arteriolar dilation to acetylcholine after transfusion of a cell-free polymeric hemoglobin oxygen carrier. The role of constitutive HO2 in the pial arteriolar dilatory response to acetylcholine was determined by using 1) HO2-null mice (HO2-/-), 2) the HO inhibitor tin protoporphyrin IX (SnPPIX), and 3) 4,5,6,7-tetrabromobenzotriazole (TBB), an inhibitor of casein kinase-2 (CK2)-dependent phosphorylation of HO2. In anesthetized mice, superfusion of a cranial window with SnPPIX decreased arteriolar dilation produced by 10 microM acetylcholine by 51%. After partial polymeric hemoglobin exchange transfusion, the acetylcholine response was normal but was reduced 72% by SnPPIX and 95% by TBB. In HO2-/- mice, the acetylcholine response was modestly reduced by 14% compared with control mice and was unaffected by SnPPIX. After hemoglobin transfusion in HO2-/- mice, acetylcholine responses were also unaffected by SnPPIX and TBB. In contrast, nitric oxide synthase inhibition completely blocked the acetylcholine responses in hemoglobin-transfused HO2-/- mice. We conclude 1) that HO2 activity partially contributes to acetylcholine-induced pial arteriolar dilation in mice, 2) that this contribution is augmented in the presence of a plasma-based hemoglobin polymer and appears to depend on a CK2 kinase mechanism, 3) that nitric oxide synthase activity rather than HO1 activity contributes to the acetylcholine reactivity in HO2-/- mice, and 4) that plasma-based polymeric hemoglobin does not scavenge all of the nitric oxide generated by cerebrovascular acetylcholine stimulation.     

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.     

Dependence of acetylcholine and ADP dilation of pial arterioles on heme oxygenase after transfusion of cell-free polymeric hemoglobin

Polymers of cell-free hemoglobin have been designed for clinical use as oxygen carriers, but limited information is available regarding their effects on vascular regulation. We tested the hypothesis that the contribution of heme oxygenase (HO) to acetylcholine-evoked dilation of pial arterioles is upregulated 2 days after polymeric hemoglobin transfusion. Dilator responses to acetylcholine measured by intravital microscopy in anesthetized cats were blocked by superfusion of the HO inhibitor tin protoporphyrin-IX (SnPPIX) in a group that had undergone exchange transfusion with hemoglobin 2 days earlier but not in surgical sham and albumin-transfused groups. However, immunoblots from cortical brain homogenates did not reveal changes in expression of the inducible isoform HO1 or the constitutive isoform HO2 in the hemoglobin-transfused group. To test whether the inhibitory effect of SnPPIX was present acutely after hemoglobin transfusion, responses were measured within an hour of completion of the exchange transfusion. In control and albumin-transfused groups, acetylcholine responses were unaffected by SnPPIX but were blocked by addition of the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine (l-NNA) to the superfusate. In hemoglobin-transfused groups, the acetylcholine response was blocked by either SnPPIX or l-NNA alone. The effect of another HO inhibitor, chromium mesoporphyrin (CrMP), was tested on ADP, another endothelial-dependent dilator, in anesthetized rats. Pial arteriolar dilation to ADP was unaffected by CrMP in controls but was attenuated 62% by CrMP in rats transfused with hemoglobin. It is concluded that 1) polymeric hemoglobin transfusion acutely upregulates the contribution of HO to acetylcholine-induced dilation of pial arterioles in cats, 2) this upregulation persists 2 days after transfusion when 95% of the hemoglobin is cleared from the circulation, and 3) this acute upregulation of HO signaling is ubiquitous in that similar effects were observed with a different endothelial-dependent agonist (i.e., ADP) in a another species (rat).     

Effects of trigeminal neurotransmitters on piglet pial arterioles.

We investigated effects of calcitonin gene-related peptide (CGRP), substance P (SP), and neurokinin A (NKA) on pial arterioles in newborn pigs. Pial arteriolar diameter was determined using a closed cranial window and intravital microscopy. Initial diameters were approximately 100 microns. Calcitonin-gene related peptide dilated pial arterioles by 22 +/- 8% at 10(-9)M and by 34 +/- 6% at 10(-8)M (n = 8), and this response was not significantly altered by prior administration of indomethacin (5mg/kg, iv) (n = 6) or administration of NG-methyl-L-arginine (5mg/kg, iv, and 10(-3)M in CSF) (n = 10). Substance P dilated arterioles at 10(-10)M through 10(-5)M (maximal response = 23 +/- 3%) (n = 6), and this response was unaffected by indomethacin administration (n = 6). In contrast, NG-methyl-L-arginine blocked much of the pial arteriolar dilation to SP. Unlike the other two peptides, NKA did not change pial arteriolar diameter. Radioimmunoassay determinations indicated that cerebrospinal fluid levels of 6-keto-prostaglandin F1 and prostaglandin E2 did not change appreciably during application of CGRP or SP. We conclude that CGRP and SP but not NKA are dilator stimuli in the piglet pial circulation. Dilation by CGRP probably involves direct activation of receptors on vascular smooth muscle, while SP probably partially dilates pial arterioles via release of an endothelium-dependent relaxing factor.     

Insensitivity of cerebral oxygen transport to oxygen affinity of hemoglobin-based oxygen carriers

The cerebrovascular effects of exchange transfusion of various cell-free hemoglobins that possess different oxygen affinities are reviewed. Reducing hematocrit by transfusion of a non-oxygen-carrying solution dilates pial arterioles on the brain surface and increases cerebral blood flow to maintain a constant bulk oxygen transport to the brain. In contrast, transfusion of hemoglobins with P50 of 4-34 Torr causes constriction of pial arterioles that offsets the decrease in blood viscosity to maintain cerebral blood flow and oxygen transport. The autoregulatory constriction is dependent on synthesis of 20-HETE from arachidonic acid. This oxygen-dependent reaction is apparently enhanced by facilitated oxygen diffusion from the red cell to the endothelium arising from increased plasma oxygen solubility in the presence of low or high-affinity hemoglobin. Exchange transfusion of recombinant hemoglobin polymers with P50 of 3 and 18 Torr reduces infarct volume from experimental stroke. Cell-free hemoglobins do not require a P50 as high as red blood cell hemoglobin to facilitate oxygen delivery.     

beta-Adrenoceptor and nNOS-derived NO interactions modulate hypoglycemic pial arteriolar dilation in rats

We examined the relative contributions from nitric oxide (NO) and catecholaminergic pathways in promoting cerebral arteriolar dilation during hypoglycemia (plasma glucose congruent with 1.4 mM). To that end, we monitored the effects of beta-adrenoceptor (beta-AR) blockade with propranolol (Pro, 1.5 mg/kg iv), neuronal nitric oxide synthase (nNOS) inhibition with 7-nitroindazole (7-NI, 40 mg/kg ip) or ARR-17477 (300 microM, via topical application), or combined intravenous Pro + 7-NI or ARR-17477 on pial arteriolar diameter changes in anesthetized rats subjected to insulin-induced hypoglycemia. Additional experiments, employing topically applied TTX (1 microM), addressed the possibility that the pial arteriolar response to hypoglycemia required neuronal transmission. Separately, Pro and 7-NI elicited modest but statistically insignificant 10-20% reductions in the normal ~40% increase in arteriolar diameter accompanying hypoglycemia. However, combined Pro-7-NI was accompanied by a >80% reduction in the hypoglycemia-induced dilation. On the other hand, the combination of intravenous Pro and topical ARR-17477 did not affect the hypoglycemia response. In the presence of TTX, the pial arteriolar response to hypoglycemia was lost completely. These results suggest that 1) beta-ARs and nNOS-derived NO interact in contributing to hypoglycemia-induced pial arteriolar dilation; 2) the interaction does not occur in the vicinity of the arteriole; and 3) the vasodilating signal is transmitted via a neuronal pathway.     

Adenosine receptors mediate glutamate-evoked arteriolar dilation in the rat cerebral cortex

We tested the hypothesis that adenosine (Ado) mediates glutamate-induced vasodilation in the cerebral cortex by monitoring pial arteriole diameter in chloralose-anesthetized rats equipped with closed cranial windows. Topical application of 100 microM glutamate and 100 microM N-methyl-d-aspartate (NMDA) dilated pial arterioles (baseline diameter 25 +/- 2 microm) by 17 +/- 1% and 18 +/- 4%, respectively. Coapplication of the nonselective Ado receptor antagonist theophylline (Theo; 10 microM) significantly reduced glutamate- and NMDA-induced vasodilation to 4 +/- 2% (P < 0.01) and 6 +/- 2% (P < 0.05), whereas the Ado A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.1 microM) had no effect. Moreover, application of the Ado A(2A) receptor-selective antagonist 4-(2-[7-amino-2-(2-furyl)(1,2,4)triazolo(2,3-a)(1,3,5)triazin-5-ylamino]ethyl)phenol (ZM-241385), either by superfusion (0.1 microM, 1 microM) or intravenously (1 mg/kg), significantly inhibited the pial arteriole dilation response to glutamate. Neither Theo nor ZM-241385 affected vascular reactivity to mild hypercapnia induced by 5% CO(2) inhalation. These results suggest that Ado contributes to the dilation of rat cerebral arterioles induced by exogenous glutamate, and that the Ado A(2A) receptor subtype may be involved in this dilation response.     

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.     

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.     

Impaired nitric oxide synthase-dependent dilatation of cerebral arterioles in type II diabetic rats

The goals of this study were to determine the effects of type II diabetes mellitus on nitric oxide synthase-dependent responses of cerebral arterioles and on endothelial nitric oxide synthase (eNOS) protein in cerebral arterioles. We examined dilatation of cerebral (pial) arterioles in 13-15 week old male lean and diabetic obese Zucker rats in response to nitric oxide synthase-dependent agonists (acetylcholine and adenosine diphosphate (ADP)) and a nitric oxide synthase-independent agonist (nitroglycerin). We found that acetylcholine (10 microM) increased cerebral arteriolar diameter by 10 +/- 3% (mean +/- SE) in lean Zucker rats, but by only 2 +/- 2% in diabetic obese Zucker rats (p<0.05). In addition, ADP (100 microM) increased cerebral arteriolar diameter by 20 +/- 2% in lean Zucker rats, but by only 8 +/- 2% in diabetic obese Zucker rats (p<0.05). In contrast, nitroglycerin produced similar vasodilatation in lean and diabetic obese Zucker rats. Thus, impaired dilatation of cerebral arterioles in diabetic obese Zucker rats is not related to non-specific impairment of vasodilatation. Following these functional studies, we harvested cerebral microvessels for Western blot analysis of eNOS protein. We found that eNOS protein was significantly higher in diabetic obese Zucker rats than in lean Zucker rats (p<0.05). Thus, type II diabetes mellitus impairs nitric oxide synthase-dependent responses of cerebral arterioles. In addition, eNOS protein from cerebral blood vessels is increased in diabetic obese Zucker rats.     

Impact of myocardial structure and function postinfarction on diastolic strain measurements: implications for assessment of myocardial viability

Venular control of arteriolar perfusion has been the focus of several investigations in recent years. This study investigated 1) whether endogenous adenosine helps control venule-dependent arteriolar dilation and 2) whether venular leukocyte adherence limits this response via an oxidant-dependent mechanism in which nitric oxide (NO) levels are decreased. Intravital microscopy was used to assess changes in arteriolar diameters and NO levels in rat mesentery. The average resting diameter of arterioles (27.5 +/- 1.0 microm) paired with venules with minimal leukocyte adherence (2.1 +/- 0.3 per 100-microm length) was significantly larger than that of unpaired arterioles (24.5 +/- 0.8 microm) and arterioles (23.3 +/- 1.3 microm) paired with venules with higher leukocyte adherence (9.0 +/- 0.5 per 100-microm length). Local superfusion of adenosine deaminase (ADA) induced significant decreases in diameter and perivascular NO concentration in arterioles closely paired to venules with minimal leukocyte adherence. However, ADA had little effect on arterioles closely paired to venules with high leukocyte adherence or on unpaired arterioles. To determine whether the attenuated response to ADA for the high-adherence group was oxidant dependent, the responses were also observed in arterioles treated with 10(-4) M Tempol. In the high-adherence group, Tempol fully restored NO levels to those of the low-adherence group; however, the ADA-induced constriction remained attenuated, suggesting a possible role for an oxidant-independent vasoconstrictor released from the inflamed venules. These findings suggest that adenosine- and venule-dependent dilation of paired arterioles may be mediated, in part, by NO and inhibited by venular leukocyte adherence.     

Role of nitric oxide scavenging in vascular response to cell-free hemoglobin transfusion

Modified Hb solutions have been developed as O(2) carrier transfusion fluids, but of concern is the possibility that increased scavenging of nitric oxide (NO) within the plasma will alter vascular reactivity even if the Hb does not readily extravasate. The effect of decreasing hematocrit from approximately 30% to 18% by an exchange transfusion of a 6% sebacyl cross-linked tetrameric Hb solution on the diameter of pial arterioles possessing tight endothelial junctions was examined through a cranial window in anesthetized cats with and without a NO synthase (NOS) inhibitor. Superfusion of a NOS inhibitor decreased diameter, and subsequent Hb transfusion produced additional constriction that was not different from Hb transfusion alone but was different from the dilation observed by exchange transfusion of an albumin solution after NOS inhibition. In contrast, abluminal application of the cross-linked Hb produced constriction that was attenuated by the NOS inhibitor. Neither abluminal nor intraluminal cross-linked Hb interfered with pial arteriolar dilation to cromakalim, an activator of ATP-sensitive potassium channels. Pial vascular reactivity to hypocapnia and hypercapnia was unaffected by Hb transfusion. Microsphere-determined regional blood flow indicated selective decreases in perfusion after Hb transfusion in the kidney, small intestine, and neurohypophysis, which does not have tight endothelial junctions. Administration of a NOS inhibitor to reduce the basal level of NO available for scavenging before Hb transfusion prevented further decreases in blood flow to these regions compared with NOS inhibition alone. In contrast, blood flow to skeletal and left ventricular muscle increased, and cerebral blood flow was unchanged after Hb transfusion. This cross-linked Hb tetramer is known to appear in renal lymph but not in urine. We conclude that cell-free tetrameric Hb does not scavenge sufficient NO in the plasma space to significantly affect baseline tone in vascular beds with tight endothelial junctions but does produce substantial constriction in beds with porous endothelium. The data support increasing the molecular size of Hb by polymerization or conjugation to limit extravasation in all vascular beds to preserve normal vascular reactivity.     

VEGF-induced relaxation of pulmonary arteries is mediated by endothelial cytochrome P-450 hydroxylase

The cytochrome P-450 metabolite 20-HETE induces calcium-, endothelial-, and nitric oxide (NO)-dependent relaxation of bovine pulmonary arteries (PA). VEGF is an NO-dependent dilator of systemic arteries and plays a key role in maintaining the integrity of the pulmonary vasculature. We tested the effect of VEGF on PA diameter and tone and the contribution of cytochrome P-450 family 4 (CYP4) to vasoactive effects of VEGF. Bovine PA rings (1 mm in diameter) relaxed with VEGF (0.1-10 nM) in an endothelial- and eNOS-dependent manner. This response was blunted by pretreatment with the CYP4 inhibitor dibromododecynyl methyl sulfonamide (DDMS) as well as a mechanistically different CYP4 inhibitor N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine. PAs also increased in diameter by 6-12% in the presence of VEGF (10 nM), and this increase was attenuated by DDMS. In contrast to that shown in PAs, 20-HETE constricted bovine renal arteries and did not increase intracellular Ca(2+) in renal artery endothelial cells as observed in bovine pulmonary artery endothelial cells (BPAECs). VEGF-evoked increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) in BPAECs were blunted by treatment with DDMS. Both VEGF (10 nM) and 20-HETE (1-5 microM) stimulated NO release from cultured BPAECs, and once again VEGF-induced increases were attenuated by pretreating the cells with DDMS. We conclude that CYP4/20-HETE contributes to VEGF-stimulated NO release and vasodilation in bovine PAs. Given the unique expression of 20-HETE-forming CYP4 in BPAECs vs. systemic arterial endothelial cells, CYP4 may be an important mediator of endothelial-dependent vasoreactivity in PAs.     

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.     

Permissive contributions of NO and prostacyclin in CO-induced cerebrovascular dilation in piglets

Endogenously produced CO is an important dilator in newborn cerebrovascular circulation. CO dilates cerebral arterioles by activating Ca2+-activated K+ channels, but modulatory actions of other effectors and second messenger inputs are unclear. Specifically, the mechanisms behind the obligatory permissive roles of prostacyclin and NO are uncertain. Therefore, the present study was performed using acutely implanted, closed cranial windows in newborn pigs to address the hypothesis that the permissive roles of NO and prostacyclin in cerebrovascular dilation in response to CO involve a common mechanism. The NO donor sodium nitroprusside restored dilation in response to CO after inhibition of that dilation with the prostaglandin cyclooxygenase inhibitor indomethacin. The stable prostacyclin analog iloprost restored CO-induced dilation blocked by the NO synthase inhibitor Nomega-nitro-L-arginine. Restoration of dilation in response to CO by the cGMP-dependent phosphodiesterase inhibitor zaprinast and blockade of CO dilation by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazole-[4,3-a]quinoxalin-1-one (ODQ) suggests involvement of the cGMP/PKG pathway. Iloprost or the cAMP-dependent dilator isoproterenol restored dilation in response to CO after ODQ administration. However, CO-induced dilation blocked by the cGMP-dependent PKG inhibitor Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine could not be reversed by administration of sodium nitroprusside, iloprost, or isoproterenol. Conversely, PKA inhibition did not block dilation in response to CO. Overall, data indicate that activation of PKG is the predominant mechanism of the permissive actions of NO and prostacyclin for CO-induced pial arteriolar dilation.