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We investigated in cerebral penetrating arterioles the signaling mechanisms and dose-dependency of extracellular magnesium-induced vasodilation and also its vasodilatory effects in vessels preconstricted with agonists associated with delayed cerebral vasospasm following SAH. Male rat penetrating arterioles were cannulated. Their internal diameters were monitored. To investigate mechanisms of magnesium-induced vasodilation, inhibitors of endothelial function, potassium channels and endothelial impairment were tested. To simulate cerebral vasospasm we applied several spasmogenic agonists. Increased extracellular magnesium concentration produced concentration-dependent vasodilation, which was partially attenuated by non-specific calcium-sensitive potassium channel inhibitor tetraethylammonium, but not by other potassium channel inhibitors. Neither the nitric oxide synthase inhibitor L-NNA nor endothelial impairment induced by air embolism reduced the dilation. Although the magnesium-induced vasodilation was slightly attenuated by the spasmogen ET-1, neither application of PF2α nor TXA2 analog effect the vasodilation. Magnesium induced a concentration- and smooth muscle cell-dependent dilation in cerebral penetrating arterioles. Calcium-sensitive potassium channels of smooth muscle cells may play a key role in magnesium-induced vasodilation. Magnesium also dilated endothelium-impaired vessels as well as vessels preconstricted with spasmogenic agonists. These results provide a fundamental background for the clinical use of magnesium, especially in treatment against delayed cerebral ischemia or vasospasm following SAH., (Copyright © 2015 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.)

Background: Substantial evidence suggests that amyloid-β (Aβ) species induce oxidative stress and cerebrovascular (CV) dysfunction in Alzheimer's disease (AD), potentially contributing to the progressive dementia of this disease. The upstream molecular pathways governing this process, however, are poorly understood. In this report, we examine the role of heparan sulfate proteoglycans (HSPG) in Aβ-induced vascular smooth muscle cell (VSMC) dysfunction in vitro., Results: Our results demonstrate that pharmacological depletion of HSPG (by enzymatic degradation with active, but not heat-inactivated, heparinase) in primary human cerebral and transformed rat VSMC mitigates Aβ(1-40⁻) and Aβ(1-42⁻)induced oxidative stress. This inhibitory effect is specific for HSPG depletion and does not occur with pharmacological depletion of other glycosaminoglycan (GAG) family members. We also found that Aβ(1-40) (but not Aβ(1-42)) causes a hypercontractile phenotype in transformed rat cerebral VSMC that likely results from a HSPG-mediated augmentation in intracellular Ca(2+) activity, as both Aβ(1-40⁻)induced VSMC hypercontractility and increased Ca(2+) influx are inhibited by pharmacological HSPG depletion. Moreover, chelation of extracellular Ca(2+) with ethylene glycol tetraacetic acid (EGTA) does not prevent the production of Aβ(1-40⁻) or Aβ(1-42⁻)mediated reactive oxygen species (ROS), suggesting that Aβ-induced ROS and VSMC hypercontractility occur through different molecular pathways., Conclusions: Taken together, our data indicate that HSPG are critical mediators of Aβ-induced oxidative stress and Aβ(1-40⁻)induced VSMC dysfunction.

Cerebral amyloid angiopathy (CAA) is characterized by deposition of amyloid β peptide (Aβ) within walls of cerebral arteries and is an important cause of intracerebral hemorrhage, ischemic stroke, and cognitive dysfunction in elderly patients with and without Alzheimer's Disease (AD). NADPH oxidase-derived oxidative stress plays a key role in soluble Aβ-induced vessel dysfunction, but the mechanisms by which insoluble Aβ in the form of CAA causes cerebrovascular (CV) dysfunction are not clear. Here, we demonstrate evidence that reactive oxygen species (ROS) and, in particular, NADPH oxidase-derived ROS are a key mediator of CAA-induced CV deficits. First, the NADPH oxidase inhibitor, apocynin, and the nonspecific ROS scavenger, tempol, are shown to reduce oxidative stress and improve CV reactivity in aged Tg2576 mice. Second, the observed improvement in CV function is attributed both to a reduction in CAA formation and a decrease in CAA-induced vasomotor impairment. Third, anti-ROS therapy attenuates CAA-related microhemorrhage. A potential mechanism by which ROS contribute to CAA pathogenesis is also identified because apocynin substantially reduces expression levels of ApoE-a factor known to promote CAA formation. In total, these data indicate that ROS are a key contributor to CAA formation, CAA-induced vessel dysfunction, and CAA-related microhemorrhage. Thus, ROS and, in particular, NADPH oxidase-derived ROS are a promising therapeutic target for patients with CAA and AD.

Objective: Cerebral vessels, such as intracerebral perforating arterioles isolated from rat brain, have been widely used as an ex vivo model to study the cerebrovascular function associated with cerebrovascular disorders and the therapeutic effects of various pharmacological agents. These perforating arterioles, however, have demonstrated differences in the vascular architecture and reactivity compared with a larger leptomeningeal artery which has been commonly implicated in cerebrovascular disease. In this study, therefore, we developed the method for studying cerebrovascular function utilizing the olfactory artery isolated from the mouse brain., Methods: The olfactory artery (OA) was isolated from the C57/BL6 wild-type mouse brain. After removing connective tissues, one side of the isolated vessel segment (approximately -500 µm in length) was cannulated and the opposite end of the vessel was completely sealed while being viewed with an inverted microscope. After verifying the absence of pressure leakage, we examined the vascular reactivity to various vasoactive agents under the fixed intravascular pressure (60 mm Hg)., Results: We found that the isolated mouse OAs were able to constrict in response to vasoconstrictors, including KCl, phenylephrine, endothelin-1, and prostaglandin PGH2. Moreover, this isolated vessel demonstrated vasodilation in a dose-dependent manner when vasodilatory agents, acetylcholine and bradykinin, were applied., Conclusion: Our findings suggest that the isolated olfactory artery would provide as a useful ex vivo model to study the molecular and cellular mechanisms of vascular function underlying cerebrovascular disorders and the direct effects of such disease-modifying pathways on cerebrovascular function utilizing pharmacological agents and genetically modified mouse models.

Elastin (Eln) insufficiency in mice and humans is associated with hypertension and altered structure and mechanical properties of large arteries. However, it is not known to what extent functional or structural changes in resistance arteries contribute to the elevated blood pressure that is characteristic of Eln insufficiency. Here, we investigated how Eln insufficiency affects the structure and function of the resistance vasculature. A functional profile of resistance vasculature in Eln(+/-) mice was generated by assessing small mesenteric artery (MA) contractile and vasodilatory responses to vasoactive agents. We found that Eln haploinsufficiency had a modest effect on phenylephrine-induced vasoconstriction, whereas ANG II-evoked vasoconstriction was markedly increased. Blockade of ANG II type 2 receptors with PD-123319 or modulation of Rho kinase activity with the inhibitor Y-27632 attenuated the augmented vasoconstriction, whereas acute Y-27632 administration normalized blood pressure in Eln(+/-) mice. Sodium nitroprusside- and isoproterenol-induced vasodilatation were normal, whereas ACh-induced vasodilatation was severely impaired in Eln(+/-) MAs. Histologically, the number of smooth muscle layers did not change in Eln(+/-) MAs; however, an additional discontinuous layer of Eln appeared between the smooth muscle layers that was absent in wild-type arteries. We conclude that high blood pressure arising from Eln insufficiency is due partly to permanent changes in vascular tone as a result of increased sensitivity of the resistance vasculature to circulating ANG II and to impaired vasodilatory mechanisms arising from endothelial dysfunction characterized by impaired endothelium-dependent vasodilatation. Eln insufficiency causes augmented ANG II-induced vasoconstriction in part through a novel mechanism that facilitates contraction evoked by ANG II type 2 receptors and altered G protein signaling.

Background: KATP channels, assembled from pore-forming (Kir6.1 or Kir6.2) and regulatory (SUR1 or SUR2) subunits, link metabolism to excitability. Loss of Kir6.2 results in hypoglycemia and hyperinsulinemia, whereas loss of Kir6.1 causes Prinzmetal angina-like symptoms in mice. Conversely, overactivity of Kir6.2 induces neonatal diabetes in mice and humans, but consequences of Kir6.1 overactivity are unknown., Methods and Results: We generated transgenic mice expressing wild-type (WT), ATP-insensitive Kir6.1 [Gly343Asp] (GD), and ATP-insensitive Kir6.1 [Gly343Asp,Gln53Arg] (GD-QR) subunits, under Cre-recombinase control. Expression was induced in smooth muscle cells by crossing with smooth muscle myosin heavy chain promoter-driven tamoxifen-inducible Cre-recombinase (SMMHC-Cre-ER) mice. Three weeks after tamoxifen induction, we assessed blood pressure in anesthetized and conscious animals, as well as contractility of mesenteric artery smooth muscle and KATP currents in isolated mesenteric artery myocytes. Both systolic and diastolic blood pressures were significantly reduced in GD and GD-QR mice but normal in mice expressing the WT transgene and elevated in Kir6.1 knockout mice as well as in mice expressing dominant-negative Kir6.1 [AAA] in smooth muscle. Contractile response of isolated GD-QR mesenteric arteries was blunted relative to WT controls, but nitroprusside relaxation was unaffected. Basal KATP conductance and pinacidil-activated conductance were elevated in GD but not in WT myocytes., Conclusions: KATP overactivity in vascular muscle can lead directly to reduced vascular contractility and lower blood pressure. We predict that gain of vascular KATP function in humans would lead to a chronic vasodilatory phenotype, as indeed has recently been demonstrated in Cantu syndrome.

Background and Purpose: Reduced risk and severity of stroke in adult females are thought to depend on normal levels of endogenous estrogen, which is a known neuro- and vasoprotective agent in experimental cerebral ischemia. Recently, a novel G protein-coupled estrogen receptor (GPER, formerly GPR30) has been identified and may mediate the vasomotor and -protective effects of estrogen. However, the signaling mechanisms associated with GPER in the cerebral microcirculation remain unclear. We investigated the mechanism of GPER-mediated vasoreactivity and also its vasoprotective effect after hypoxia/reoxygenation (H/RO) injury., Methods: Rat cerebral penetrating arterioles from both sexes were isolated, cannulated, and pressurized. Vessel diameters were recorded by computer-aided videomicroscopy. To investigate vasomotor mechanism of the GPER agonist (G-1), several inhibitors with or without endothelial impairment were tested. Ischemia/reperfusion injury was simulated using H/RO. Vasomotor responses to adenosine triphophate after H/RO were measured with or without G-1 and compared with controls., Results: G-1 produced a vasodilatory response, which was partially dependent on endothelium-derived nitric oxide (NO) but not arachidonic acid cascades and endothelial hyperpolarization factor. Attenuation of G-1-vasodilation by the NO synthase inhibitor and endothelium-impairment were greater in vessels from female than male animals. G-1 treatment after H/RO injury fully restored arteriolar dilation to adenosine triphophate compared with controls., Conclusions: GPER agonist elicited dilation, which was partially caused by endothelial NO pathway and induced by direct relaxation of smooth muscle cells. Further, GPER agonist restored vessel function of arterioles after H/RO injury and may play an important role in the ability of estrogen to protect the cerebrovasculature against ischemia/reperfusion injury.

Endocrine disrupting chemicals with estrogenic activity play an important role as obesogens. However, studies investigating the most potent natural estrogen, estradiol-17β (E2), at low dose are lacking. We examined endocrine and physiological parameters in gilts receiving distinct concentrations of E2 during pregnancy. We then investigated whether adverse effects prevail in progeny due to a potential endocrine disruption. E2 was orally applied to gilts during the entire period of pregnancy. The concentrations represented a daily consumption at the recommended ADI level (0.05 μg/kg body weight/day), at the NOEL (10 μg/kg body weight/day) and at a high dosage (1000 μg/kg body weight/day). Plasma hormone concentrations were determined using enzyme immuno assays. Offspring body fat was assessed by dual-energy X-ray absorptiometry scanning. In treated gilts receiving 1000μg E2/kg body weight/day we found significantly elevated plasma E2 levels during pregnancy, paralleled by an increased weight gain. While offspring showed similar weight at birth, piglets exhibited a significant reduction in weight at weaning even though their mothers had only received 0.05 μg E2/kg body weight/day. At 8 weeks of age, specifically males showed a significant increase in overall body fat percentage. In conclusion, prenatal exposure to low doses of E2 affected pig offspring development in terms of body weight and composition. In line with findings from other obesogens, our data suggest a programming effect during pregnancy for E2 causative for the depicted phenotypes. Therefore, E2 exposure may imply a possible contribution to childhood obesity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objective: VaD is the second-most common form of dementia, second only to that caused by AD. As the name indicates, VaD is predominantly considered a disease caused by vascular phenomena., Methods: In this invited review, we introduce the reader to recent developments in defining VaD as a unique form of dementia by reviewing the current pertinent literature. We discuss the clinical and experimental evidence that supports the notion that the microcirculation, specifically cell-to-cell communication, likely contributes to the development of VaD. Through exploration of the concept of the NVU, we elucidate the extensive cerebrovascular communication that exists and highlight models that may help test the contribution(s) of cell-to-cell communication at the microvascular level to the development and progression of VaD. Lastly, we explore the possibility that some dementia, generally considered to be purely neurodegenerative, may actually have a vascular component at the neurovascular level., Conclusion: This latter recognition potentially broadens the critical involvement of microvascular events that contribute to the numerous dementias affecting an increasingly larger sector of the adult population., (© 2012 John Wiley & Sons Ltd.)

Regulator of G protein signaling 2 (RGS2) is a GTPase-activating protein for G(q/11)α and G(i/o)α subunits. RGS2 deficiency is linked to hypertension in mice and humans, although causative mechanisms are not understood. Because endothelial dysfunction and increased peripheral resistance are hallmarks of hypertension, determining whether RGS2 regulates microvascular reactivity may reveal mechanisms relevant to cardiovascular disease. Here we have determined the effects of systemic versus endothelium- or vascular smooth muscle-specific deletion of RGS2 on microvascular contraction and relaxation. Contraction and relaxation of mesenteric resistance arteries were analyzed in response to phenylephrine, sodium nitroprusside, or acetylcholine with or without inhibitors of nitric oxide (NO) synthase or K(+) channels that mediate endothelium-derived hyperpolarizing factor (EDHF)-dependent relaxation. The results showed that deleting RGS2 in vascular smooth muscle had minor effects. Systemic or endothelium-specific deletion of RGS2 strikingly inhibited acetylcholine-evoked relaxation. Endothelium-specific deletion of RGS2 had little effect on NO-dependent relaxation but markedly impaired EDHF-dependent relaxation. Acute, inducible deletion of RGS2 in endothelium did not affect blood pressure significantly. Impaired EDHF-mediated vasodilatation was rescued by blocking G(i/o)α activation with pertussis toxin. These findings indicated that systemic or endothelium-specific RGS2 deficiency causes endothelial dysfunction resulting in impaired EDHF-dependent vasodilatation. RGS2 deficiency enables endothelial G(i/o) activity to inhibit EDHF-dependent relaxation, whereas RGS2 sufficiency facilitates EDHF-evoked relaxation by squelching endothelial G(i/o) activity. Mutation or down-regulation of RGS2 in hypertension patients therefore may contribute to endothelial dysfunction and defective EDHF-dependent relaxation. Blunting G(i/o) signaling might improve endothelial function in such patients.

Activation of phospholipases leads to the release of arachidonic acid and lysophospholipids that play prominent roles in regulating vasomotor tone. To identify the role of calcium-independent phospholipase A(2)beta (iPLA(2)beta) in vasomotor function, we measured vascular responses to phenylephrine (PE) and ACh in mesenteric arterioles from wild-type (WT; iPLA(2)beta(+/+)) mice and those lacking the beta-isoform (iPLA(2)beta(-/-)) both ex vivo and in vivo. Vessels isolated from iPLA(2)beta(-/-) mice demonstrated increased constriction to PE, despite lower basal smooth muscle calcium levels, and decreased vasodilation to ACh compared with iPLA(2)beta(+/+) mice. PE constriction resulted in initial intracellular calcium release with subsequent steady-state constriction that depended on extracellular calcium influx. Endothelial denudation had no effect on vessel tone or PE-induced constriction although the dilation to ACh was significantly reduced in iPLA(2)beta(+/+) vessels. In contrast, vessels from iPLA(2)beta(-/-) constricted by 54% after denudation, indicating smooth muscle hypercontractility. In vivo, blood pressure, resting vessel diameter, and constriction of mesenteric vessels to PE were not different in iPLA(2)beta(-/-) vessels compared with WT mouse vessels. However, relaxation after ACh administration in situ was attenuated, indicating an endothelial inability to induce dilation in response to ACh. In cultured endothelial cells, inhibition of iPLA(2)beta with (S)-(E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL) decreased endothelial nitric oxide synthase phosphorylation and reduced endothelial agonist-induced intracellular calcium release as well as extracellular calcium influx. We conclude that iPLA(2)beta is an important mediator of vascular relaxation and intracellular calcium homeostasis in both smooth muscle and endothelial cells and that ablation of iPLA(2)beta causes agonist-induced smooth muscle hypercontractility and reduced agonist-induced endothelial dilation.

Background: Evidence indicates that soluble forms of amyloid-beta (Abeta) are vasoactive, which may contribute to cerebrovascular dysfunction noted in patients with Alzheimer's Disease and cerebral amyloid angiopathy. The effects of soluble Abeta on penetrating cerebral arterioles - the vessels most responsible for controlling cerebrovascular resistance - have not been studied., Results: Freshly dissolved Abeta1-40 and Abeta1-42, but not the reverse peptide Abeta40-1 constricted isolated rat penetrating arterioles and diminished dilation to adenosine tri-phosphate (ATP). Abeta1-42 also enhanced ATP-induced vessel constriction. Abeta1-40 diminished arteriolar myogenic response, and an anti-Abeta antibody reduced Abeta1-40 induced arteriolar constriction. Prolonged Abeta exposure in vessels of Tg2576 mice resulted in a marked age-dependent effect on ATP-induced vascular responses. Vessels from 6 month old Tg2576 mice had reduced vascular responses whereas these were absent from 12 month old animals. Abeta1-40 and Abeta1-42 acutely increased production of reactive oxygen species (ROS) in cultured rat cerebro-microvascular cells. The radical scavenger MnTBAP attenuated this Abeta-induced oxidative stress and Abeta1-40-induced constriction in rat arterioles., Conclusions: Our results suggest that soluble Abeta1-40 and Abeta1-42 directly affect the vasomotor regulation of isolated rodent penetrating arterioles, and that ROS partially mediate these effects. Once insoluble Abeta deposits are present, arteriolar reactivity is greatly diminished.

Through oxygen-dependent release of the vasodilator ATP, the mobile erythrocyte plays a fundamental role in matching microvascular oxygen supply with local tissue oxygen demand. Signal transduction within the erythrocyte and microvessels as well as feedback mechanisms controlling ATP release have been described. Our understanding of the impact of this novel control mechanism will rely on the integration of in vivo experiments and computational models.

Background: Adenosine triphosphate (ATP), a potent vascular regulator in the cerebral circulation, initiates conducted vasomotor responses which may be impaired after pathological insults. We analyzed the mechanism of ATP-induced local vasomotor responses and their effect on conducted vasomotor responses in rat cerebral penetrating arterioles., Methods: Arterioles were cannulated and their internal diameter monitored. Vasomotor responses to ATP were observed in the presence or absence of inhibitors, or after endothelial impairment. Smooth muscle membrane potentials were measured in some vessels., Results: Microapplication of ATP produced a biphasic response (constriction followed by dilation), which resulted in conducted dilation preceded by a membrane hyperpolarization. alpha,beta-methylene-ATP or pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) blunted the ATP-mediated constriction and enhanced local and conducted dilation. N(omega)-monomethyl-L-arginine, endothelial impairment and N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH) reduced the local dilation caused by ATP. The conducted dilation was attenuated by MS-PPOH and endothelial impairment, but not N(omega)-monomethyl-L-arginine or indomethacin., Conclusion: ATP-induced conducted dilation is preceded by membrane hyperpolarization. Local ATP induces initial local constriction via smooth-muscle P(2X1) and subsequent dilation via endothelial P(2Y) receptors. Nitric oxide, cytochrome P450 metabolites, and intermediate and large conductance K(Ca) channels mediate dilation caused by ATP. ATP-induced conducted dilation is dependent upon both the endothelium and cytochrome P450 metabolites., (Copyright (c) 2008 S. Karger AG, Basel.)

The contributing effect of cerebrovascular pathology in Alzheimer's disease (AD) has become increasingly appreciated. Recent evidence suggests that amyloid-beta peptide (Abeta), the same peptide found in neuritic plaques of AD, may play a role via its vasoactive properties. Several studies have examined young Tg2576 mice expressing mutant amyloid precursor protein (APP) and having elevated levels of soluble Abeta but no cerebral amyloid angiopathy (CAA). These studies suggest but do not prove that soluble Abeta can significantly impair the cerebral circulation. Other studies examining older Tg2576 mice having extensive CAA found even greater cerebrovascular dysfunction, suggesting that CAA is likely to further impair vascular function. Herein, we examined vasodilatory responses in young and older Tg2576 mice to further assess the roles of soluble and insoluble Abeta on vessel function. We found that (1) vascular impairment was present in both young and older Tg2576 mice; (2) a strong correlation between CAA severity and vessel reactivity exists; (3) a surprisingly small amount of CAA led to marked reduction or complete loss of vessel function; 4) CAA-induced vasomotor impairment resulted from dysfunction rather than loss or disruption of vascular smooth muscle cells; and 5) acute depletion of Abeta improved vessel function in young and to a lesser degree older Tg2576 mice. These results strongly suggest that both soluble and insoluble Abeta cause cerebrovascular dysfunction, that mechanisms other than Abeta-induced alteration in vessel integrity are responsible, and that anti-Abeta therapy may have beneficial vascular effects in addition to positive effects on parenchymal amyloid.

Background and Purpose: P2 receptors are important regulators of cerebrovascular tone. However, there is functional heterogeneity of P2Y receptors along the vascular tree, and the functionality of P2Y receptors in small arterioles has not been studied in detail. We investigated the effects of activating P2Y1 and P2Y2 receptors and their underlying dilator mechanisms in rat intracerebral arterioles., Methods: We used computer-aided videomicroscopy to measure diameter responses from isolated and pressurized rat penetrating arterioles (39.9+/-1.2 microm) to the natural P2 receptor agonist ATP in addition to ADP-beta-S (P2Y1-selective) and ATP-gamma-S (P2Y2-selective) and inhibitors of signaling pathways., Results: Extraluminal application of ATP-gamma-S and ADP-beta-S initiated a biphasic response (initial constriction followed by the secondary dilation) similar to ATP-induced responses. Pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (0.1 mmol/L; a P2Y1 receptor antagonist) blocked ADP-beta-S- but not ATP-gamma-S-induced dilation and affected the ATP-mediated dilation at low concentrations. Nomega-Monomethyl-l-arginine partially inhibited the dilation of ATP and ADP-beta-S but not ATP-gamma-S. High K+ saline suppressed the dilation of all agonists. Indomethacin had no effect., Conclusions: Both P2Y1 and P2Y2 receptors are functionally present in cerebral arterioles. ATP stimulates P2Y1 receptors at low concentrations, while high concentrations of ATP activate P2Y2 in addition to P2Y1 receptors. Nitric oxide is involved in P2Y1 but not P2Y2 receptor activation. Potassium channels play an important role in the regulation of P2Y receptor-mediated dilation.

Background and Purpose: Extracellular concentration of potassium ion ([K+]o) may have a significant influence on the cerebral circulation in health and disease. Mechanisms of [K+]o-induced conducted vasomotor responses in cerebral arterioles, possibly linking microvascular regulation to neuronal activity, have not been examined., Methods: We analyzed vascular responses to small increases of [K+]o (up to 5 mmol/L) in isolated, cannulated, and pressurized rat cerebral arterioles (36.5+/-1.4 micro m). [K+]o was elevated globally through extraluminal application or locally through micropipette, while arteriolar diameter was measured online., Results: Elevation of [K+]o (5 mmol/L) produced dilation that was inhibited by ouabain but not BaCl2. Locally applied [K+]o (3 to 5 mmol/L) produced a biphasic response (initial constriction followed by dilation), both of which were conducted to the remote site (distance 1142+/-68 microm). Endothelial impairment inhibited conducted but not local biphasic responses. Extraluminal ouabain attenuated local and conducted secondary dilation but not initial constriction. The local biphasic response was unaffected by extraluminal or intraluminal BaCl2. Extraluminal but not intraluminal BaCl2 impaired both conducted constriction and dilation., Conclusions: In rat penetrating arteriole, (1) [K+]o (3 to 5 mmol/L) strongly regulates arteriolar tone and causes conducted vasomotor responses; (2) local responses to elevated [K+]o are endothelium independent but conducted responses are dependent on an intact endothelium; (3) smooth muscle Na+-K+-ATPase activation is the generator of conducted dilation; and (4) smooth muscle inward rectifier potassium channels sustain conduction. Our findings suggest that potassium-induced conducted vasomotor responses may link local neuronal activity to microvascular regulation, which may be attenuated in pathological conditions.

Background and Purpose: Potassium channels or nitric oxide or both are major mediators of acidosis-induced dilation in the cerebral circulation. However, these contributions depend on a variety of factors such as species and vessel location. The present study was designed to clarify whether potassium channels and endothelial nitric oxide are involved in acidosis-induced dilation of isolated rat cerebral arterioles., Methods: Cerebral arterioles were cannulated and monitored with an inverted microscope. Acidosis (pH 6.8 to 7.4) produced by adding hydrogen ions mediated dilation of the cerebral arterioles in a concentration-dependent manner. The role of nitric oxide and potassium channels in response to acidosis was examined with several specific inhibitors and endothelial damage., Results: The dilation was significantly inhibited by potassium chloride (30 mmol/L) and glibenclamide (3 micromol/L; ATP-sensitive potassium channel inhibitor). We found that 30 micromol/L BaCl2 (concentration-dependent potassium channel inhibitor) also affected the dilation; however, an additional treatment of 3 micromol/L glibenclamide did not produce further inhibition. Tetraethylammonium ion (1 mmol/L; calcium-activated potassium channel inhibitor) and 4-aminopyridine (100 micromol/L; voltage-dependent potassium channel inhibitor) as well as ouabain (10 micromol/L; Na-K ATPase inhibitor) and N-methylsulphonyl-6-(2-proparglyloxyphenyl) hexanamide (1 micromol/L; cytochrome P450 epoxygenase inhibitor) did not alter acidotic dilation. N(omega)-Monomethyl-L-arginine (10 micromol/L) and N(omega)-nitro-L-arginine (10 micromol/L) as nitric oxide synthase inhibitor blunted the dilation. Furthermore, the dilation was significantly attenuated after the endothelial impairment. Additional treatment with glibenclamide (3 micromol/L) further reduced the dilation in response to acidosis., Conclusions: Endothelial nitric oxide and smooth muscle ATP-sensitive potassium channels contribute to acidosis-induced dilation of rat cerebral arterioles. Endothelial damage caused by pathological conditions such as subarachnoid hemorrhage or traumatic brain injury may contribute to reduced blood flow despite injury-induced cerebral acidosis.

Basic fibroblast growth factor (bFGF), a potent mitogen, acutely dilates cerebral blood vessels and may be effective in reducing cerebral infarction. However, the vasodilatory mechanism, which may involve nitric oxide (NO), is not completely understood. This study investigated whether membrane hyperpolarization is also involved in this mechanism. Membrane potential (MP) of smooth muscle cells and vessel diameter of isolated intracerebral arterioles were simultaneously measured following extraluminal application of bFGF in rats. The involvement of NO and adenosine triphosphate-sensitive potassium (KATP) channels in bFGF-induced vasodilation and membrane hyperpolarization was evaluated using specific inhibitors, NG-monomethyl-L-arginine (L-NMMA, 10(-4) M) and glibenclamide (GB, 10(-5) M), respectively. The resting MP was recorded at a mean value of -31.9 +/- 4.5 mV. bFGF (1 to 1000 ng/ml) produced significant vasodilation and hyperpolarization. Treatment with L-NMMA caused vasoconstriction and significantly attenuated bFGF-induced vasodilation without affecting membrane hyperpolarization. In the presence of GB, the membrane potential was significantly depolarized but the vessel diameter was only marginally reduced, so bFGF-induced membrane hyperpolarization was inhibited while arteriolar dilation was attenuated. These results suggest that bFGF-induced vasodilation is mediated by a mechanism involving both NO and membrane hyperpolarization, and that membrane hyperpolarization is caused by the activation of KATP channels.

Effects of extraluminal UTP were studied and compared with vascular responses to ATP and its analogs in rat cerebral-penetrating arterioles. UTP, UDP, 2-methylthio-ATP, and alpha,beta-methylene-ATP dilated arterioles at the lowest concentration and constricted them at high concentrations. Low concentrations of ATP dilated the vessels; high concentrations caused a biphasic response, with transient constriction followed by dilation. Endothelial impairment inhibited ATP- and UTP-mediated dilation and potentiated constriction to UTP but not to ATP. ATP- and 2-methylthio-ATP- but not UTP-mediated constrictions were inhibited by desensitization with 10(-6) M alpha,beta-methylene-ATP or 3 x 10(-6) M pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). PPADS at 10(-4) M abolished the UTP-mediated constriction and induced vasodilation in a dose-dependent manner but did not affect the dilation to ATP. These results suggest that in rat cerebral microvessels 1) ATP and 2-methylthio-ATP induce transient constriction via smooth muscle P(2X1) receptors in the cerebral arteriole, 2) UTP stimulates two different classes of P(2Y) receptors, resulting in constriction (smooth muscle P(2Y4)) and dilation (possibly endothelial P(2Y2)), and 3) ATP and UTP produce dilation by stimulation of a single receptor (P(2Y2)).

Background and Purpose: Potassium channels are important regulators of resting tone in large cerebral arteries, but their activity and distribution may vary according to vessel location and species studied. In the cerebral microcirculation in vivo, however, these channels appear to be silent at rest. Our goal was to determine the activity of potassium channels of brain arterioles from 2 origins under basal conditions in vitro., Methods: Penetrating cerebral (40. 9+/-2.2 microm control diameter) and brain stem (36.2+/-1.2 microm) arterioles of rats were prepared from middle cerebral and basilar arteries, respectively. The internal diameter of cannulated and pressurized vessel was monitored with the inverted microscope before and after administration of potassium channel inhibitors. In addition, we studied the effect of nitric oxide synthase inhibition on potassium channel activity., Results: Cerebral and brain stem arterioles were significantly constricted by 4-aminopyridine and low concentration of BaCl(2) but not by glibenclamide. The addition of N:(omega)-nitro-L-arginine to 4-aminopyridine further decreased diameters of both arterioles. Tetraethylammonium ion caused a significant constriction of brain stem but not cerebral arteriole. The brain stem arteriole was further constricted by additional N:(omega)-nitro-L-arginine., Conclusions: Voltage-dependent and inward-rectifier, but not ATP-sensitive, potassium channels are active under basal conditions of rat cerebral and brain stem arterioles. There is a regional difference in the activity of calcium-activated potassium channels, which, at rest, are open in brain stem but silent in cerebral arterioles. In addition, basal endogenous nitric oxide may not contribute to the activation of voltage-dependent and calcium-activated potassium channels.

The matching of blood flow with metabolic need requires a mechanism for sensing the needs of the tissue and communicating that need to the arterioles, the ultimate controllers of tissue perfusion. Despite significant strides in our understanding of blood flow regulation, the identity of the O(2) sensor has remained elusive. Recently, the red blood cell, the Hb-containing O(2) carrier, has been implicated as a potential O(2) sensor and contributor to this vascular control by virtue of its concomitant carriage of millimolar amounts of ATP, which it is able to release when exposed to a low-O(2) environment. To evaluate this possibility, we exposed perfused cerebral arterioles to low extraluminal O(2) in the absence and presence of red blood cells or 6% dextran and determined both vessel diameter and ATP in the vessel effluent. Only when the vessels were perfused with red blood cells did the vessels dilate in response to low extraluminal O(2). In addition, this response was accompanied by a significant increase in vessel effluent ATP. These findings support the hypothesis that the red blood cell itself serves a role in determining O(2) supply to tissue.

The mechanisms responsible for subarachnoid hemorrhage (SAH)-induced vasospasm are under intense investigation but remain incompletely understood. A consequence of SAH-induced vasospasm, cerebral infarction, produces a nonrecoverable ischemic tissue core surrounded by a potentially amenable penumbra. However, successful treatment has been inconsistent. In this review, we summarize the basic molecular biology of cerebrovascular regulation, describe recent developments in molecular biology to elucidate the mechanisms of SAH-induced vasospasm, and discuss the potential contribution of cerebral microcirculation regulation to the control of ischemia. Our understanding of the pathogenesis of SAH-induced vasospasm remains a major scientific challenge; however, molecular biological techniques are beginning to uncover the intracellular mechanisms involved in vascular regulation and its failure. Recent findings of microvascular regulatory mechanisms and their failure after SAH suggest a role in the development and size of the ischemia. Progress is being made in identifying the various components in the blood that cause SAH-induced vasospasm. Thus, our evolving understanding of the underlying molecular mechanism may provide the basis for improved treatment after SAH-induced vasospasm, especially at the level of the microcirculation.

Objective: Cerebral autoregulation is an important regulatory mechanism that maintains a constant cerebral blood flow over a wide range of perfusion pressures. The goal of this study was to determine whether nitric oxide contributes to the autoregulatory response of cerebral arterioles to altered transmural pressure (TMP)., Methods: Seventy-nine intraparenchymal arterioles (53.6 +/- 3.5 microm mean diameter) isolated from rats were cannulated with micropipettes and pressurized at a TMP of 60 mm Hg (control pressure). Vessel diameters were monitored continuously using a video dimensional analyzer. The autoregulatory diameter responses to varying intraluminal pressures were observed in the presence and absence of a nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA). The effect of L-NMMA-induced constriction on autoregulatory response also was compared with responses after prostaglandin F2alpha and alkalosis-induced constrictions., Results: Autoregulatory responses were observed over a range from 10 to 90 mm Hg of TMP. Treatment with 10(-4) mol/L L-NMMA constricted arterioles and inhibited the autoregulatory vasodilation to TMP reductions from 60 mm Hg to 10 or 30 mm Hg. In L-NMMA-treated arterioles, elevation in TMP from 60 to 90 mm Hg caused an autoregulatory vasoconstriction. Treatment with alkaline pH 7.65 constricted arterioles to a similar degree as that induced by L-NMMA at 60 mm Hg, and under these conditions, the autoregulatory response remained intact. Arterioles severely constricted with prostaglandin F2alpha showed no significant autoregulatory response., Conclusion: These results suggest that 1) vascular nitric oxide release increases in response to a decrease in TMP from 60 mm Hg, thereby contributing to the autoregulatory vasodilation intrinsic to the vessel during hypotension, 2) arteriolar nitric oxide appears not to be involved in the autoregulatory vasoconstriction induced by elevating TMP from 60 to 90 mm Hg, and 3) a marked increase in vascular tone may affect autoregulatory response.

After subarachnoid hemorrhage (SAH), cerebral arteries display impaired vasomotor control, resulting in decreased regional cerebral blood flow. Recently, propagation of vasomotor responses has been recognized as an important regulatory mechanism in microcirculation. In this study, the authors tested the hypothesis that oxyhemoglobin (OxyHb) inhibits the vasodilatory effect of chemical mediators such as adenosine and adenine nucleotides at a local and/or propagated site. Penetrating intracerebral arterioles were surgically isolated from the middle cerebral arteries of rat brains, cannulated, and observed videomicroscopically in an organ bath under an inverted microscope. The effects of 10(-5) M OxyHb on vasoactive responses to adenosine, adenosine diphosphate (ADP), and adenosine triphosphate (ATP) were examined. The drugs were extraluminally applied either to the bath (10(-10)-10(-3) M) or, using pressure microejection (pipette concentration 10(-2) M), locally. The ATP and ADP initially constricted and then significantly dilated the vessels after both extraluminal application and microapplication. Furthermore, local microstimulation by these drugs produced conducted vasodilation. Adenosine elicited significant vasodilation after both extraluminal and local stimulation. Again, conducted vasodilation was observed. The vasomotor responses that were induced by a maximum local stimulation corresponded in magnitude to those observed at bath concentrations of 10(-5) to 10(-4) M of the same drug. Pretreatment with OxyHb constricted arterioles to an average of 87% of control and blunted extraluminally induced dilation at low concentrations (10(-10)-10(-8)) of ATP and ADP, but did not affect vasodilation induced by 10(-4) M or greater concentrations of ATP, ADP, or adenosine. Although the local response to local microstimulation was unaltered, propagated vasodilation as a response to ATP, ADP, and adenosine was significantly attenuated by OxyHb. These findings indicate that vasodilatory propagation plays an important role in the regulation of brain microcirculation and that its impairment by OxyHb could, in part, explain the cerebral hypoperfusion that is observed after SAH.

We tested the hypothesis that conduction of vasomotor responses occurs in cannulated and isolated rat cerebral penetrating arterioles. Both at the site of stimulation (local) and 500-650 microns distant from it, we observed the diameter responses and time courses thereof to pressure-ejected vasoactive stimuli. ATP locally caused an initial constriction (response onset at 0.3 s, average diameter 85% of control at 450-ms pulse with a maximum at 1.6 s after stimulation) followed by a secondary dilation (111% at 7 s). Conducted vasodilation of 111% was observed over a distance of 520 microns. Prostaglandin F2 alpha (PGF2 alpha) constricted the vessels locally (80%) and caused conducted vasodilation (110%). For both ATP and PGF2 alpha the local constriction occurred simultaneously to the conducted vasodilation. Adenosine dilated the vessels (123%) but produced only inconsistent conducted vasodilation. Hydrogen ions initially constricted the vessels (88%) and then dilated them to 113%. Thus, although ATP and PGF2 alpha are strong promoters of conduction, adenosine and hydrogen ions are not. Paradoxically, ATP and PGF2 alpha caused conducted vasodilation even though the initial local response was a vasoconstriction, indicating that in cerebral arterioles conduction may be mediated through endothelial cell mechanisms rather than through smooth muscle cell communication.

Local regulation of microvascular blood flow is a complex process in which the needs of the tissue must be communicated to the vasculature, enabling the appropriate matching of O2 supply to demand. We hypothesize that the red blood cell is not only the major O2 carrier but also serves as an O2 sensor and affecter of changes in O2 delivery via its release of ATP, which subsequently binds to P2y receptors on the vascular endothelium, altering vessel caliber. Using the hamster as a model, we determined that the efflux of ATP from red blood cells after exposure to low-PO2 (PO2 = 17 +/- 6 mmHg) and low-pH (pH = 7.06 +/- 0.07) solutions was significantly (P < 0.01) greater than that after exposure to normoxic, normal pH (PO2 = 87 +/- 4; pH = 7.38 +/- 0.04) solutions, indicating that two factors that are associated with an impaired O2 supply relative to demand increase the release of ATP from the red blood cell. To ascertain whether ATP alters vascular caliber, we applied 10(-6) M ATP intraluminally to arterioles of the retractor muscle, using a micropressure system. Vessel diameter increased 8 and 10%, 140 +/- 60 microns upstream of the site of infusion after 50- and 500-ms pulses, respectively. Application of ATP to arteriolar and venular capillaries induced a 31 and 81% increase in red blood cell supply rate, respectively. These results support our hypothesis that the red blood cell is more than just an O2 carrier and has a direct role in the regulation of vascular tone.

Purpose: To evaluate the size and shape of commercially available polyvinyl alcohol (PVA) particles and to determine whether they change in size when suspended in nonionic contrast and in a solution of nonionic contrast and absolute alcohol., Methods: The two-dimensional area and the long and short axis of PVA particles from several different vendors were measured using a light microscope attached to a video system and an image-processing software program. Particles were measured as packaged (dry or suspended in saline), suspended in ioversol, and suspended in ioversol containing 30% alcohol., Results: All brands of dry particles had similar microscopic appearances. The saline-suspended particles had fewer and finer perforations. After suspension in contrast, all sizes and brands of dry particles significantly increased in size. The particles packaged in saline did not expand. The addition of alcohol to the contrast did not consistently change particle size. Particle aggregation was similar in both contrast suspensions for all groups of particles. Particles less than 50 microns in size were rarely observed in any PVA preparation after suspension., Conclusions: The three dry PVA preparations seem to be similar. All increase significantly in size when suspended in nonionic contrast or contrast-alcohol solutions. The saline-packaged PVA particles were different from the dry variety and did not enlarge in contrast or contrast-alcohol solutions. Alcohol did not change the size or suspension characteristics of PVA particles. Particles less than 50 microns in size were rarely identified.

Background and Purpose: Although cerebral penetrating arterioles are main regulators of the brain microcirculation, little is known about the effect of endothelium-derived relaxation factor on these vessels. This study examined the effects of nitric oxide synthase inhibitors on the spontaneous tone of isolated rat cerebral arterioles., Methods: Intraparenchymal penetrating arterioles (53 to 102 microns in passive diameter) isolated from Sprague-Dawley rats were cannulated with glass pipettes and subjected to 60 mm Hg of intraluminal pressure. The diameter response to intraluminal and extraluminal treatments was observed with an inverted microscope., Results: Extraluminal application of Nw-nitro-L-arginine (10(-5) mol/L) contracted the arterioles to 63.9 +/- 2.8% (P < .05) of the control diameter. This contracting effect was stereospecific and easily reversed by L-arginine dose dependently (10(-3), 10(-2) mol/L) but not by D-arginine. Intraluminally applied Nw-nitro-L-arginine also induced a similar degree of contraction. Another nitric oxide synthase inhibitor, NG-monomethyl L-arginine (10(-5), 10(-4) mol/L), applied extraluminally induced a dose-dependent contraction to 77.5 +/- 6.6% and 68.6 +/- 5.4% of the control (P < .05), which was also reversed by L-arginine. L-Arginine alone did not significantly affect vessel diameter, however. Treatment with indomethacin, a cyclooxygenase inhibitor, dilated the vessel to 115.2 +/- 7% (P < .05) but did not change the constricting effect of Nw-nitro-L-arginine., Conclusions: Nw-Nitro-L-arginine and NG-monomethyl L-arginine produce substantial contraction in isolated brain arterioles, suggesting that nitric oxide of brain arterioles is continuously produced within the vessel wall. The dilatory effect of indomethacin appears to be independent of the vasoconstriction induced by nitric oxide synthase inhibitor. In these vessels, the effect of nitric oxide synthase inhibitors is not mediated by an indomethacin-sensitive mechanism. A balance probably exists between factors tending to constrict these arterioles and the elaboration of nitric oxide from endothelial cells, which tends to dilate them. The production of nitric oxide from isolated vessels indicates that parenchymal and vessel wall sources of nitric oxide are probably important in brain microcirculatory regulation.

The relationship between cell membrane potential, vessel diameter, and pH in small cerebral arterioles is not completely understood. This study involved direct, simultaneous measurement of cell membrane potential and vessel diameter at various extracellular pH levels. Arterioles ranging from 44 to 91 microns in diameter were isolated, transferred to a temperature-controlled microscope chamber, which was used as an organ bath, and observed through an inverted videomicroscope. Two vessel cannulation procedures were used: a single-sided cannulation with the other side occluded, and a double-sided and perfused cannulation. After cannulation, the vessels were pressurized to 60 mm Hg intraluminally and the bath temperature was raised to 37 degrees C. Cell membrane potentials of vessel wall cells were obtained after the bath temperature reached 37 degrees C with the vessels partly constricted and again after spontaneous tone (constriction) of the healthy vessels had developed. The effect of extraluminal pH on cell membrane potentials was studied by changing the bath pH from 7.3 to either 7.65 or 6.8 in the single-sided cannulation. The average cell membrane potential for vessels at 37 degrees C, with 60 mm Hg of intraluminal pressure and pH 7.3, was -37.5 mV. The cell membrane potential depolarized to -30.9 mV at pH 7.65 and hyperpolarized to -58.4 mV at pH 6.8, with a slope of 25.8 mV/pH unit. The effect of depolarizing extracellular potassium ions on the cell membrane potential was examined by perfusing two vessels with modified Ringer's solution containing 70 mM KCl. This perfusion method decreased the vessel diameter by 48% and depolarized the observed cell membrane potential from -41.9 to -19.8 mV, with a slope of -0.42 mV per percentage diameter change. These data provide the first measurements of membrane potentials of isolated penetrating arteriole wall cells in vitro. The results indicate that the cell membrane potential relates linearly to the vessel diameter. This new technique opens the possibility for studying vessel response to stimuli under controlled conditions and regulatory mechanisms such as the propagation of vasomotor responses.

The coupling between intracellular Ca2+ concentration ([Ca2+]i) and smooth muscle cell (SMC) contractility is well known, but recent reports suggest that SMCs can contract without an increase in [Ca2+]i. Penetrating arterioles were isolated from rat cortex, cannulated, and loaded extraluminally with fura 2. We used ratio imaging of corresponding vessel wall areas to estimate and correlate [Ca2+]i to the vessel diameter during various extraluminal treatments. Control diameter was 45.6 +/- 3.9 (SE) microns with an estimated [Ca2+]i of 181 +/- 18 nM. Extraluminal papaverine or pH of 6.8 dilated the vessels to 61.0 +/- 6.6 and 57.8 +/- 5.8 microns and decreased [Ca2+]i to 108 +/- 33 and 155 +/- 8 nM, respectively. Alkaline pH of 7.65 or the Ca2+ ionophore ionomycin constricted the arterioles to 34.8 +/- 3.9 and 30.0 +/- 5.8 microns and increased [Ca2+]i to 273 +/- 47 and 853 +/- 155 nM, respectively. These results show an inverse relationship between vessel diameter and [Ca2+]i. Blocking the production of endothelium-derived nitric oxide (EDNO) with N omega-nitro-L-arginine constricted the vessels to 34.4 +/- 3.5 microns without raising but lowering [Ca2+]i to 157 +/- 44 nM. The dissociation of vessel tone and estimated [Ca2+]i after EDNO blocking has not been reported before in cerebral arterioles and may indicate that EDNO regulates vascular tone in a Ca(2+)-independent manner.

Video microscopy of red cell flow in capillaries at the surface of skeletal muscle provided the opportunity to quantitate ischemia-reperfusion (I-R) induced microcirculatory changes, in vivo. Extensor Digitorum Longus (EDL) muscles of 22 male Wistar rats (300-400 g), anesthetized with sodium pentobarbital (Somnotol, 65 mg kg,-1 IP), were used to measure the number of perfused capillaries (CDper: mm-1) crossing lines drawn perpendicular to the muscle axis, and red blood cell velocity (VRBC: mm/s) within individual capillaries from controls (n = 6), and after 2 hr (n = 4), 3 hr (n = 4), and 4 hr (n = 5) of no-flow ischemia with the muscle temperature maintained at its normal value of 32 degrees C. Ischemia was induced by tightening a tourniquet placed around the limb above the EDL muscle. Measurements were made after 30, 60, and 90 min of reperfusion. To test the usefulness of this skeletal muscle model for evaluating proposed interventions in I-R, the effect of hypothermia (24 degrees C) on the microcirculation following 4 hr ischemia (n = 3) was measured. Edema formation was estimated from the wet/dry weight ratio of the ischemic and contralateral control EDL muscles. Capillary perfusion at the surface of the control muscles was remarkably stable over the 5 hr period studied, while significant changes occurred following the ischemic periods. Significantly lower CDper was measured 30 min following all periods of normothermic ischemia. However, unlike the 2 and 4 hr ischemic periods 3 hr normothermic ischemia resulted in a progressive decline in CDper throughout the reperfusion period. VRBC showed evidence of a hyperemic response following 2 hr normothermic ischemia (control: 0.12 mm/s +/- 0.19 compared to 0.26 mm/s +/- 0.03 following 90 min reperfusion; mean +/- sem). However, no such hyperemia was measured following either 3 or 4 hr normothermic ischemia (i.e., 3 hr control: 0.24 mm/s +/- 0.01 compared to 0.07 mm s +/- 0.003 following 90 min reperfusion). In fact, VRBC was essentially zero 90 min following 4 hr normothermic ischemia (0.01 mm/s +/- 0.01). However, when the muscle was allowed to cool to 24 degrees C during 4 hr ischemia no significant change in either VRBC or CDper was measured compared to pre-ischemic controls. Evidence of edema was found after 3 and 4 hr normothermic ischemia. This study establishes a skeletal muscle model of I-R, which may be useful in testing hypotheses regarding mechanisms of I-R injury, and effectiveness of proposed treatments of I-R.

We have developed a new method for quantification of arteriolar hydraulic conductivity (Lp) from isolated rat brain vessels. The volume flux of water per unit surface area across the arteriole wall (Jv/S) was assessed from measurements of silicon oil drop movement within an occluded vessel at two to three pressures (between 20 and 70 mmHg); the Lp was derived from the slope of the relationship between Jv/S and applied pressure. Lp was measured in isolated cerebral arterioles 1) at room temperature (22 degrees C) without spontaneous vessel tone (control Lp; n = 11), 2) at room temperature with 10(-4) M adenosine (n = 5), and 3) at 37 degrees C with vessels dilated submaximally with 10(-4) M adenosine (n = 6). Lp at 22 degrees C without adenosine was 13.2 +/- 4.2 x 10(-9) (+/- SE) cm.s-1.cmH2O-1 for all vessels studied. Lp values ranged from 1.2 to 44.1 x 10(-9) cm.s-1.cmH2O-1 with a median value that was 5.9 x 10(-9) cm.s-1.cmH2O-1. Lp increased significantly (on average, 2.6-fold) with adenosine at 37 degrees C but not with adenosine at 22 degrees C. Control Lp bore no relationship to either the development of spontaneous tone or the diameter response to pH change, two recognized indicators of vessel viability.

Recently, Dietrich (1989, Microvasc. Res. 38, 125-135) demonstrated that a local application of a minute amount of norepinephrine (NE, 5.5 mM, 0.01-88 pmole) on a capillary in rat mesentery can elicit constriction of the feeding arteriole 0.5-1.0 mm away. This constriction can reduce or even stop blood flow in capillaries supplied by the arteriole. The main objective here was to show that the phenomenon of reduced flow occurs not only in the rat mesentery but also in other tissues and species. We chose to study the rat tibialis anterior and frog sartorius muscles. Using the same intravital video-microscopic approach as in the mesentery, strong NE stimuli (3 mM) were applied iontophoretically 48 times to 19 capillaries in 10 rats anesthetized with pentobarbital. They resulted in significant reductions (average: 80%) of the red blood cell velocity (VRBC) in capillaries. The onset of these reductions (i.e., 10% decrease from control) occurred within 3-52 sec (average: 20.9 sec) from the time of NE application. Reductions lasted 6.0 min. The same stimuli were applied 42 times to 15 capillaries in 6 frogs anesthetized with urethane. The average VRBC reduction was 86%. The onset occurred within 30.6 sec while the reduction lasted 6.6 min. Under the same conditions, arteriolar diameters in the sartorius muscle decreased significantly from 28.5 to 22.5 microns (n = 8). We also used local microinjection of small droplets of NE (30 mM) to 13 capillaries in 7 frogs. This resulted in a significant VRBC reduction of 64% with an onset time of 44.2 sec and a reduction duration of 17.2 min. Weak NE stimuli (3 microM) applied iontophoretically to 10 capillaries in 5 frogs resulted in marginal, but significant, VRBC reductions (9%). The present study demonstrates that the phenomenon of reduced flow after local application of NE may be a general phenomenon as it occurs also in skeletal muscle in both rat and frog. Our accompanying paper addresses the hypothesis that the phenomenon reflects communication of a NE-induced signal along the capillary.

The preceding study (Dietrich and Tyml, 1992. Microvasc. Res. 43) demonstrated that a local application of norepinephrine (NE) on a capillary in a skeletal muscle produces a temporary reduction in blood flow within this capillary. The reduction is mediated via constriction of the supplying arteriole. The objective of the present study was to address the mechanism by which the local NE stimulus is propagated from the capillary to the arteriole. Using intravital video microscopy we measured red blood cell velocity in capillaries, and diameter of supplying arterioles, in the sartorius muscle in anesthetized frogs. Velocity responses were measured following iontophoretic application of NE (3 mM in the pipette) on the capillary, with or without pretreatment with 0.9 mM tetrodotoxin (nerve-specific sodium channel blocker), 30 mM lidocaine (nonspecific sodium channel blocker), and 30 mM yohimbine (alpha 2-receptor blocker). Diameter responses were measured before and after capillary damage introduced by microcautery. Tetrodotoxin did not block the NE-induced velocity reduction (i.e., from 0.2 to 0.07 mm/sec), while lidocaine attenuated it. Yohimbine blocked it only when applied on the same site as NE. Capillary damage abolished the NE-induced arteriolar constriction (i.e., from 27.8 to 21.5 microns). We conclude that the observed responses were not due to (1) direct diffusion of NE from the capillary to the arteriole, (2) conduction along adrenergic nerves, or (3) venous-arteriolar diffusional cross-talk. We interpret our data to indicate that the capillary itself could function as a communicating medium.

Epinephrine (5.5 mM; quantity: 0.01-87 pmole) and norepinephrine (5.9 mM; quantity: 0.01-88 pmole) were applied to 144 and 26 mesenteric capillaries, respectively, in 39 rats. Neither of these agents had a significant effect on the mean capillary diameter (control diameter before epinephrine application: 9.6 +/- 3.6 microns SD; before norepinephrine application: 11.4 +/- 6.6 microns SD). Unexpectedly, in 36% of the epinephrine applications and 49% of the norepinephrine applications, capillary blood flow was either reduced (epinephrine: within 6-61 sec; norepinephrine: within 3-36 sec) or stopped completely (epinephrine: within 8-124 sec; norepinephrine: 11-124 sec). These reductions (duration: 12 sec-32 min for epinephrine; 21 sec-12 min for norepinephrine) were associated with flow reductions in all neighboring capillaries fed by a common supplying arteriole. In order to check the possible direct effect of drug diffusion norepinephrine was applied at increasing distances up to 400 microns from the arteriole. For all such distances arterioles constricted much quicker than predicted by diffusion. Fluorescent staining using glyoxylic acid showed the presence of adrenergic nerves around arterioles, along capillaries as well as in the avascular tissue. It is concluded that conduction along nerves, or possibly via endothelial cell-to-cell communication, could explain the intriguing retrograde effect of locally applied drugs on the supplying arteriole.

An attempt should be made to summarize the history of ophthalmology in a small series of postage stamps. Eighteen stamps could be dedicated to themes recalling the achievements of famous ophthalmologists. In this way it would be possible to acquaint the public with some medical history while at the same time encouraging an interesting leisure pastime.

In-vivo studies were carried out on frog mesentery in order to determine the precise and specific actions of micropipette application of adrenaline on arterioles, precapillary sphincters and capillaries. A technique was employed, using liquid paraffin and micropipettes to deliver droplets to a particular microvessel; the droplets varied between 10-20 microns in diameter. All vessel types, including segments of capillaries, demonstrated constrictor responses to the local application of adrenaline. The time course of these constrictor events varied with the vessel type, with the sphincter being the most rapid in response.