Canine Middle Cerebral Arteries Research Articles

Abstract 137: Dose-dependent Improvement In Stroke Outcomes Following Intra-arterial Mesenchymal Stem Cell Therapy In A Canine Endovascular Stroke Model

Introduction: Cell-based therapies for stroke remain under development and there is a lack of pre-clinical large animal data defining optimal dosing and delivery methods. Whereas safety and efficacy of intra-arterial (IA) mesenchymal stem cells (MSC) have been demonstrated in rodent models, large animal model are lacking, hampering advancement of the field and limiting mechanistic insights. Methods: An endovascular canine reversible Middle Cerebral Artery Occlusion (MCAO) model using a retractable platinum coil for 60-120 min was established. At 48-hour post-MCAO, allogeneic male canine MSCs (10-80 million) were delivered using a microcatheter in the ipsilateral upper cervical internal carotid artery of female canine subjects. Serial MRIs and neurological deficit scoring (NDS) were performed over 30 days. Animals were euthanized at15-30 d post-MCAO and brains were harvested for histology and molecular analyses. Results: There was a dose-dependent reduction of infarct volume on MRI associated with improved neurological scoring and corticospinal tract caliber on Diffusion Tensor Imaging over the range of 10, 20 and 40 million MSCs vs. PBS control. This dose-range was safe and did not result in worsening of stroke volume or behavioral outcomes. In contrast, 80 million IA MSCs led to a worsened neurological score immediately following the injection and an increase in the infarct volume at 4 days post injection. Conclusion: Together these findings establish a successful preclinical canine model of stroke in which dose-dependent efficacy of IA MSC therapy is demonstrated to reduce stroke size and augment DTI. These findings have important clinical implications in designing early-stage trials of IA delivery in humans.

Abstract 45: Vwf Inhibitor Lyses Middle Cerebral Artery Occlusion After 6 Hours Of Large Vessel Occlusion Stroke

Introduction: Acute ischemic stroke (AIS) is the leading cause of combined morbidity and mortality worldwide. Recombinant tissue plasminogen activator (rtPA) is the only FDA-approved drug to treat AIS, however, it is limited to treating patients within 4.5 hours of stroke onset because of the risk of intracranial hemorrhage. This limits rtPA administration to ~6% of patients. Moreover, it poorly lyses large vessel occlusion (LVO) stroke. Endovascular mechanical thrombectomy (MT) effectively recanalizes LVO but is limited to highly specialized hospitals, leaving the majority without acute therapy. Hypothesis: We hypothesize that targeted von Willebrand Factor (VWF) inhibition by DTRI-031 will recanalize arterial thrombosis in a canine model of LVO stroke, and DTRI-031 activity will be rapidly reversed by DTRI-025, which is specifically designed to inhibit DTRI-031. Methods: Utilizing a canine embolic middle cerebral artery occlusion (eMCAO) model of LVO stroke, we assessed DTRI-031 on vessel recanalization after 6 hours of LVO stroke by digital subtraction angiography (DSA). We also measured the effect of recanalization on infarct volume (efficacy) and hemorrhage (safety) using magnetic resonance imaging (MRI). Finally, we assessed the ability of DTRI-025, an oligonucleotide that binds DTRI-031 to reverse DTRI-031 activity. Results: DTRI-031 administration after 6 hours of LVO stroke resulted in ≥TICI 2A in 62.5% and ≥TICI 2B in 50% of canines (n=8). The negative control group demonstrated no revascularization (n=7). Recanalization resulted in reduced infarct volume compared to the negative control (p<0.05, unpaired t-test). None of the animals that received DTRI-031 had an intracranial hemorrhage on MRI. Finally, DTRI-025 completely reversed the activity of DTRI-031 within 5 minutes of administration in both whole blood impedance aggregometry (WBA) and PFA-100. There was no difference in heart rate, blood pressure, or temperature among the 3 groups studied. Conclusion: DTRI-031 effectively recanalizes LVO after 6 hours of stroke onset with reduced infarct volume and no incidence of hemorrhage. DTRI-025 rapidly reverses DTRI-031. This drug/reversal agent combination represents a robust yet safe approach to treat AIS.

Therapeutic effect of nerve growth factor on canine cerebral infarction evaluated by MRI.

To explore therapeutic effect of nerve growth factor (NGF) on cerebral infarction by establishing canine middle cerebral artery occlusion (MCAO) infarct model. The magnetic resonance imaging (MRI) technology was used to study effects of NGF on cerebral infarction, and the results of MRI indexes (such as diffusion-weighted imaging (DWI) and perfusion-weighted imaging (PWI)) were compared with the results of pathology, cell biology and molecular biology. The clinical manifestations of the canine infarction model treated by NGF were significantly improved within 7 days compared with control group. The therapeutic evaluation of NGF effect could be determine by canine cerebral infarction treated by NGF within 6 hours according to DWI and PWI. From 6 hours to 7 days, therapeutic evaluation of NGF could be determine by T1WI, T2WI and FLAIR. DWI and PWI could find the change of cerebral ischemia at the early stage, provide advantages for qualitative diagnosis of early-stage cerebral infarction and observation of efficacy in early treatment, initially showing that their great potential for NGF role on cerebral ischemia and mechanism.

Tetrahydrobiopterin: Mediator of Endothelial Protection

During the past twenty years, the essential role of endothelial cells in preservation of vascular homeostasis has been well established.1,2⇓ Protection of the vascular endothelium against harmful influences of circulating substances including excessive levels of lipids has been a major therapeutic approach in prevention of atherosclerosis. While endothelial dysfunction, defined as a loss of biologically active nitric oxide (NO) produced in the endothelium, has been recognized as a prime target for prevention and reversal of atherosclerotic process, identification of exact molecular mechanisms responsible for the loss of endothelial NO have been more difficult to determine. In the current issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Alp et al3 report a series of elegant studies on genetically modified mice supporting the concept that NO synthase cofactor, tetrahydrobiopterin (Figure 1), is an important vascular protective molecule. The beneficial effect of tetrahydrobiopterin in apolipoprotein E (ApoE)-deficient mice appears to be mediated by preservation of endothelial nitric oxide synthase (eNOS) activity and production of nitric oxide in endothelial cells. The major steps of tetrahydrobiopterin biosynthesis. GTP=guanosine 5′-triphosphate. See page 445 In the early 1990s it was recognized that in the presence of suboptimal concentrations of tetrahydrobiopterin, activation of purified neuronal nitric oxide synthase (nNOS) causes “uncoupling of NOS” with subsequent increase …

Mechanisms of Cerebral Arterial Relaxations to Hydrogen Peroxide

The role of hydrogen peroxide in the regulation of cerebral arterial tone is not completely understood. Previous studies have demonstrated that hydrogen peroxide causes vasodilation of small cerebral arteries. The present study was designed to determine the mechanisms responsible for relaxations of large cerebral arteries to hydrogen peroxide. Rings of canine middle cerebral arteries without endothelium were suspended for isometric force recording in modified Krebs-Ringer bicarbonate solution bubbled with 94% O(2)/6% CO(2) (37 degrees C, pH 7.4). Radioimmunoassay technique was used to determine the levels of cAMP and cGMP. During contraction to UTP (3 x 10(-6) or 10(-5) mol/L), hydrogen peroxide (10(-6) to 10(-4) mol/L) caused concentration-dependent relaxations. Catalase (1200 U/mL) abolished the relaxations to hydrogen peroxide. Inhibition of cyclooxygenase by indomethacin (10(-5) mol/L) significantly reduced relaxations to hydrogen peroxide. In arteries contracted by KCl (20 mmol/L), the relaxations to hydrogen peroxide were significantly reduced. In the presence of a nonselective potassium channel inhibitor, BaCl(2) (10(-4) mol/L), a delayed rectifier potassium channel inhibitor, 4-aminopyridine (10(-3) mol/L), or a calcium-activated potassium channel inhibitor, charybdotoxin (3 x 10(-8) mol/L), the relaxations to hydrogen peroxide were also significantly reduced. An ATP-sensitive potassium channel inhibitor, glyburide (5 x 10(-6) mol/L), did not affect the relaxations to hydrogen peroxide. Hydrogen peroxide produced concentration-dependent increase in levels of cAMP. Indomethacin (10(-5) mol/L) inhibited the stimulatory effect of hydrogen peroxide on cAMP production. In contrast, hydrogen peroxide did not affect the levels of cGMP. These results suggest that hydrogen peroxide may cause relaxations of large cerebral arteries in part by activation of arachidonic acid metabolism via cyclooxygenase pathway with subsequent increase in cAMP levels and activation of potassium channels.

Angiotensin-(1–7) causes endothelium-dependent relaxation in canine middle cerebral artery

The heptapeptide, angiotensin-(1–7), is an active member of the renin–angiotensin system. The present study was designed to characterize the role of endothelium in relaxations of large cerebral arteries to angiotensin-(1–7). Rings of canine middle cerebral arteries were suspended in organ chambers for isometric force recording. The levels of cyclic guanosine 3′,5′-monophosphate (cGMP) were assessed by radioimmunoassay. During contraction to uridine 5′-triphosphate (UTP, 3×10 −6 to 10 −5 mol/l), angiotensin-(1–7) (10 −9 to 3×10 −5 mol/l) caused concentration-dependent relaxations in arteries with endothelium, but not in endothelium-denuded vessels. Angiotensin-(1–7) significantly increased formation of cGMP. Nitric oxide synthase inhibitor, N-ω-nitro- l-arginine methyl ester ( l-NAME, 3×10 −4 mol/l), and selective soluble guanylate cyclase inhibitor, 1 H-[1,2,4]oxadiazolo[4,3- a]quinozalin-1-one (ODQ, 3×10 −6 mol/l), abolished angiotensin-(1–7)-induced relaxations. Angiotensin receptor antagonists, losartan (10 −5 mol/l), PD 123 319 (10 −5 mol/l), [Sar 1,Thr 8]-angiotensin II (10 −5 mol/l) [Sar 1,Val 5,Ala 8]-angiotensin II (10 −5 mol/l) or [7- d-Ala]-angiotensin 1–7 (10 −6 mol/l) did not affect these relaxations. However, angiotensin-converting enzyme inhibitor, captopril (10 −5 mol/l) augmented relaxations to angiotensin-(1–7). Finally, bradykinin B 2 receptor antagonist, [ d-Arg 0,Hyp 3,Thi 5, d-Tic 7,Oic 8]-bradykinin (HOE 140, 5×10 −8 mol/l) significantly reduced the effect of angiotensin-(1–7), while bradykinin B 1 receptor antagonist, des-Arg 9, [Leu 8]-bradykinin (6×10 −9 mol/l) did not influence the vascular response to the heptapeptide. These findings indicate that (1) angiotensin-(1–7) produces relaxation of canine middle cerebral arteries by the release of nitric oxide from endothelial cells, (2) angiotensin receptors do not mediate endothelium-dependent relaxations to the heptapeptide, and (3) this effect appears to be dependent on activation of local production of kinins. Our studies support the concept that angiotensin-(1–7), as a natural vasodilator hormone, may counterbalance the hemodynamic actions of angiotensin II.

Magnesium and cardiovascular disease.

Magnesium and cardiovascular disease.

The effect of 1H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ) and charybdotoxin (CTX) on relaxations of isolated cerebral arteries to nitric oxide

The mechanism underlying smooth muscle relaxations of cerebral arteries in response to nitric oxide is still not completely understood. The present study was designed to determine the role of soluble guanylate cyclase in the relaxations to a nitric oxide/nucleophile complex, diethylaminodiazen-1-ium-1,2-dioate (DEA-NONOate). Rings of canine middle cerebral arteries without endothelium were suspended in Krebs–Ringer bicarbonate solution for isometric tension recording. The levels of guanosine 3′,5′-cyclic monophosphate (cyclic GMP) were measured by radioimmunoassay technique. During contractions to uridine 5′-triphosphate (UTP), DEA-NONOate (10 −10 to 10 −5 M) caused concentration-dependent relaxations. Measurements of cyclic GMP levels in cerebral arterial wall demonstrated that DEA-NONOate is a potent stimulator of guanylate cyclase and subsequent formation of cyclic GMP. Increasing concentrations of a selective soluble guanylate cyclase inhibitor, 1H-[1,2,4]-oxadiazolo[4,3- a]quinoxalin-1-one (ODQ), caused concentration-dependent reduction of both cyclic GMP production and relaxations to DEA-NONOate. Interestingly, in the presence of the highest concentration (3×10 −6 M) of ODQ, production of cyclic GMP in response to 10 −6 M of DEA-NONOate was abolished, whereas the same concentration of DEA-NONOate caused almost complete relaxation, suggesting that mechanisms independent of cyclic GMP production may mediate relaxing effect of high concentration of a nitric oxide donor. A selective Ca 2+-activated potassium channel blocker charybdotoxin (CTX) significantly reduced relaxations to DEA-NONOate resistant to ODQ, supporting the idea that in cerebral arteries nitric oxide may activate potassium channels independently of cyclic GMP. The results of our study suggest that under physiological conditions, guanylate cyclase is a key mediator of cerebral arterial relaxations to nitric oxide. However, under pathological conditions associated with induction of nitric oxide synthase and increased biosynthesis of nitric oxide (e.g., cerebral ischemia, inflammation, sepsis), mechanisms other than formation of cyclic GMP may be activated.

Hypoxia-induced inhibition of the response to nitroxidergic nerve stimulation in canine cerebral arteries.

In isolated canine middle cerebral arteries contracted with prostaglandin F2 alpha, transmural electrical stimulation (TES), nicotine, and substance P produced relaxations. Transmural electrical stimulation- and nicotine-induced endothelium-independent responses are mediated by nitric oxide (NO) liberated from perivascular nerve, whereas substance P-induced relaxations are mediated by endothelium-derived NO. These responses were attenuated by replacement of 95% O2 and 5% CO2 gas (about 550 mm Hg of partial O2 pressure) with 95% N2 and 5% CO2 gas (about 40 mm Hg); inhibition of the response to TES was stabilized 30 minutes later. Reoxygenation partially reversed the response. Relaxations caused by exogenous NO were not influenced by hypoxia. Inhibition by hypoxia of the response to TES was not affected by superoxide dismutase. However, the inhibitory effect was prevented by amiloride and dimethyl-amiloride, Na(+)-H+ exchange inhibitors, or acidosis caused by the addition of HCl. The inhibition by hypoxia was reversed by amiloride. It is concluded that depression by hypoxia of the response mediated by endogenous NO is associated with impaired membrane function caused by restoration of normal intracellular pH by Na(+)-H+ exchanger.

Role of potassium channels in relaxations of canine middle cerebral arteries induced by nitric oxide donors.

The mechanisms underlying smooth muscle relaxations of cerebral arteries in response to nitric oxide (NO) and cyclic GMP (cGMP) are still not completely understood. The present study was designed to determine the role of potassium channels in the relaxations to NO donors 3-morpholinosydnonimine (SIN-1) and sodium nitroprusside (SNP), as well as 8-bromo-3',5' -cGMP (a synthetic analogue of cGMP) and zaprinast (a selective cGMP phosphodiesterase inhibitor). Rings of canine middle cerebral asteries without endothelium were suspended in Krebs-Ringer bicarbonate solution for isometric tension recording. The levels of cGMP were measured by radioimmunoassay. Relaxations to NO donors 8-bromo-cGMP and zaprinast were studied in the presence and in the absence of K+ channel blockers charybdotoxin (large-conductance Ca(2+)-activated K+ channels), glyburide (ATP-sensitive K+ channels), 4-aminopyridine (delayed rectifier K+ channels), and BaCl2 (multiple types of K+ channels). Concentration-dependent relaxations caused by NO donors (SIN-1 and SNP) were significantly reduced in arteries treated with BaCl2 (3 x 10(-4) mol/L) or charybdotoxin (3 x 10(-8) mol/L). Relaxations to 8-bromo-cGMP were not affected by the same concentrations of BaCl2 and charybdotoxin; however, they were reduced by higher concentrations of BaCl2 (3 x 10(-3) mol/L) or charybdotoxin (10(-7) mol/L). Zaprinast-induced relaxations were significantly reduced by BaCl2 (3 x 10(-4) mol/L) or charybdotoxin (3 x 10(-8) mol/L). Glyburide (10(-5) mol/L) and 4-aminopyridine (10(-3) mol/L) did not alter the relaxations to SIN-1 or SNP. The production of cGMP stimulated by SIN-1 in the vascular smooth muscle was not affected by BaCl2 (3 x 10(-3) mol/L) or charybdotoxin (10(-7) mol/L). These results indicate that in canine middle cerebral arteries, a significant portion of relaxations to NO liberated from nitrovasodilators is mediated by large-conductance Ca(2+)-activated K+ channels. Other K+ channels, sensitive to BaCl2, may also be involved in the mechanism of relaxations induced by NO.

Reversal of endothelin-induced vasoconstriction by endothelium-dependent and -independent vasodilators in isolated hearts and vascular rings.

Endothelin (ET-1) is a potent endogenous vasoconstrictor. Several factors increase ET-1 release in vitro and ET-1 levels increase in vivo in situations that damage blood vessels. The aim of this study was to test the activity of several differently acting vasodilator drugs on reversing or attenuating the vasoconstrictor effects of exogenously administered ET-1 in isolated guinea-pig hearts, in isolated rings with intact endothelium from canine middle cerebral and basilar arteries, and from guinea-pig aortas. Vasodilator drugs tested up to maximal concentrations were adenosine (ADE), nitroprusside (NP), acetylcholine (ACH), nifedipine (NIF), and butanedione monoxime (BDM), an excitation-uncoupling agent. Variables measured in isolated hearts included coronary flow, percentage oxygen extraction (% O2E), left ventricular pressure (LVP), and myocardial oxygen consumption. It was found that ADE, NP, ACH, and BDM each attenuated the 60% decrease in coronary flow and 20% increase in % O2E elicited by 0.5 nM ET-1 in isolated hearts, but only BDM restored coronary flow, whereas BDM and ADE both restored % O2E. In isolated rings constricted with 20 nM ET-1, BDM restored tone equivalent to that by papaverine, whereas NP and NIF only attenuated the vasoconstriction elicited by ET-1. Ring experiments also demonstrated that the vasodilatory effect of BDM was independent of nitric oxide-dependent pathways and that BDM attenuated vasoconstriction resulting from increased bath KCl. The study suggests that drugs affecting intracellular Ca2+ with a mechanism of action downstream from cell-membrane receptors or intracellular messengers may be more effective for reversing the constrictor effect of ET-1. NP, however, would be a better clinical choice for reversing ET-1-induced vasoconstriction.

Effect of tetrahydrobiopterin on endothelial function in canine middle cerebral arteries.

Tetrahydrobiopterin is an essential cofactor required for activation of NO synthase. However, in intact arteries, the exact role of tetrahydrobiopterin in the regulation of NO synthase activity is not fully understood. The present study was designed to determine the effect of increasing intracellular tetrahydrobiopterin levels on endothelial function in isolated canine middle cerebral arteries. The arterial segments were incubated in MEM for 24 hours at 37 degrees C in the presence or absence of a tetrahydrobiopterin precursor, sepiapterin (10(-4) mol/L), and/or superoxide dismutase (150 U/mL). The rings were suspended for isometric tension recording. Tetrahydrobiopterin levels were assayed by high-performance liquid chromatography. Production of cGMP was measured by radioimmunoassay. Incubation with sepiapterin markedly increased intracellular tetrahydrobiopterin levels. In sepiapterin-treated arteries, endothelium-dependent relaxations to calcium ionophore A23187 and intracellular cGMP levels were significantly reduced. Superoxide dismutase alone did not affect either relaxation to A23187 or production of cGMP. However, when arteries were incubated with superoxide dismutase plus sepiapterin, endothelium-dependent relaxations to A23187, as well as cGMP production, were significantly augmented. The augmentation of cGMP was observed in rings with (but not without) endothelium. Incubation of arteries in calcium-free medium almost abolished the synergistic effect of tetrahydrobiopterin and superoxide dismutase on cGMP production. These results demonstrate that increased availability of tetrahydrobiopterin may activate endothelial NO synthase. This effect appears to be critically dependent on the presence of superoxide dismutase.

Monthly Update: Central & Peripheral Nervous Systems: Pharmacology of antimigraine 5-HT1Dreceptor agonists

The 5-HT 1D receptor agonist sumatriptan is undoubtedly a significant advance in the management of migraine and cluster attacks. Notwithstanding, sumatriptan has some important shortcomings, which include low oral bioavailability, headache recurrence (possibly due to short t1/2) and contraindication in patients with coronary artery disease. These shortcomings, as well as lofty sales potentials, have stimulated the search and design of second-generation 5-HT1D receptor agonists. The large list of such compounds is growing, but those that are likely to be marketed in this millennium include: zolmitriptan, MK-462, naratriptan, BMS-180048 and, perhaps, the non-indole derivative alniditan. However, none of the newer 5-HT1D receptor agonists, as is also the case with sumatriptan, shows selectivity towards either the 5-HT1Dα or the 5-HT1Dβ receptor subtype and there seems to be little qualitative difference in their pharmacological profiles. Thus, these compounds suppress adenylyl cyclase activity, contract isolated cerebral (e.g., human, bovine or canine middle cerebral artery) as well as, though weakly, some peripheral arteries (e.g., human coronaries), reduce carotid artery blood flow in the cat, dog or pig by a selective action on arteriovenous anastomoses, and inhibit dural plasma extravasation (rat) and evoked potentials in dorsal horn neurones (cat) following trigeminal ganglion stimulation. Human brachial arteriovenous anastomoses are also constricted by sumatriptan. The newer 5-HT1D receptor agonists appear to offer some advantage over sumatriptan with respect to bioavailability, onset of action or headache recurrence.

Suppression of cerebral vasodilation with endothelin-1.

We investigated the effect of endothelin-1 on relaxation responses induced by vasodilator substances in canine middle cerebral arteries to better understand regulation of cerebrovascular tone and its potential impact on mechanism of cerebral vasospasm. Endothelin-1 elicited concentration-dependent contractions in helical strips of canine cerebral arteries (EC50; 4.62 x 10(-9) M). Pretreatment with 10(-9) M endothelin-1 significantly reduced endothelium-dependent relaxation elicited by substance P and endothelium-independent relaxations by nitroglycerin, prostaglandin I2, and KCl. Although endothelin-1 in a lower concentration (10(-10) M) did not affect these endothelium-independent relaxations, it did inhibit endothelium-dependent relaxation caused by substance P. A low concentration (10(-10) M) of endothelin-1 also significantly reduced endothelium-dependent relaxation of canine mesenteric arteries induced by acetylcholine. Other vasoconstrictor peptides such as angiotensin-II and vasopressin did not inhibit endothelium-dependent and -independent relaxations. These results indicate that endothelin-1 not only produces cerebral vasoconstriction but also interferes with vasodilator mechanisms and that endothelium-dependent vasodilation is more sensitive to the inhibitory effect of endothelin-1 than endothelium-independent vasodiltion.

Magnesium Sulfate and Diphenylhydantoin Relax Canine Middle Cerebral Artery Contracted with the Thromboxane-A2Analogue U46619 by Different Mechanisms: Implications for the Management of Eclampsia

Objectives: To investigate the relaxant effect of MgSO4 and diphenylhydantoin on isolated canine middle cerebral artery (MCA) rings contracted with the thromboxane-A2 analogue U46619.Methods: Canine MCA rings, with and without endothelium, were suspended in organ chambers for isometric tension recording. They were contracted with U46619 in the presence of indomethacin, with and without nitro-l-arginine (NLA). Cumulative dose-response curves were performed using MgSQ4 (10−8 M to 10−2 M) and diphenylhydantoin (10−9 M to 10−3 M).Main Outcome Measures: Tension at each concentration of the study drug expressed as a percentage of the maximum tension developed with U46619; area under the concentration response curve.Results: Both MgSO4 and diphenylhydantoin relaxed the MCA rings irrespective of the presence of endothelium. Diphenylhydantoin caused an endothelium-dependent relaxation at concentrations above 3 10−5 M, unaffected by NLA.Conclusions: MgSO4 relaxes canine MCA by a direct smooth muscle effect independ...

Effects of nitro-L-arginine on endothelium-dependent relaxation of canine cerebral arteries.

1. The effect of NG-nitro-L-arginine (NO2Arg) on the relaxation of canine basilar artery was investigated and compared with those of middle cerebral and femoral arteries. 2. NO2Arg (10(-7)-3 x 10(-5) mol/L) inhibited the substance-P (Sub-P; 10(-12)-10(-8) mol/L) induced relaxation in the basilar artery precontracted with prostaglandin F2 alpha (PGF2 alpha; 10(-5) mol/L) or KCl (10(-2) mol/L) in a concentration-dependent manner and a ratio of the maximum inhibition by NO2Arg (3 x 10(-5) mol/L) was more than 90%. 3. The relaxation induced by A23187 (10(-9)-3 x 10(-6) was also abolished by NO2Arg (3 x 10(-5) mol/L), but that by glyceryl trinitrate (GTN; 10(-9)-3 x 10(-5) mol/L) was not, in the basilar artery precontracted with PGF2 alpha (10(-5) mol/L). NG-nitro-D-arginine (NO2ArgD; 3 x 10(-5) mol/L) did not affect the relaxation induced by Sub-P (10(-12)-10(-8) mol/L). 4. L-arginine (L-Arg; 3 x 10(-5)-10(-4) mol/L) did not inhibit Sub-P (10(-12)-10(-8) mol/L) induced relaxation in the basilar artery. Pretreatment of L-Arg (10(-4) mol/L) reversed the relaxation inhibited by NO2Arg (3 x 10(-6) mol/L) in the arteries. 5. NO2Arg (3 x 10(-5) mol/L) inhibited the Sub-P (10(-12)-10(-8) mol/L) induced relaxation in the canine middle cerebral artery as much as in the basilar artery.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of cerebral arterial tone by endothelium-derived relaxing factor

This study was designed to further elucidate the role of the endothelium in regulation of cerebral vascular smooth muscle tone. Dose-dependent vasoconstrictive effects of serotonin (5-HT) were examined in endothelium-intact and endothelium-denuded ring segments prepared from canine basilar and middle cerebral arteries. Some preparations were pretreated with 10(-5) M N omega-nitro-L-arginine (L-NNA), an agent that inhibits the production of L-arginine-derived nitric oxide, one of the compounds proposed to be endothelium-derived relaxing factor. L-NNA alone elicited marked dose-dependent increases in tension in endothelium-intact preparations; a significantly smaller response was seen in endothelium-denuded preparations. The effects of L-NNA on endothelium-intact preparations were partially reversed by washing and treatment with L-arginine. The maximum tension induced by 5-HT was approximately doubled by removal of the endothelium as well as by L-NNA treatment of endothelium-intact preparations; a slight increase in maximum tension occurred in endothelium-denuded preparations treated with L-NNA. The concentration of 5-HT producing half-maximal contraction (ED50) was not affected by L-NNA. These data suggest that L-arginine-derived nitric oxide modulates canine cerebral arterial tone in both the resting state and during contraction with 5-HT.

Effect of K+ channel blockade with tetraethylammonium on anesthetic-induced relaxation in canine cerebral and coronary arteries.

The mechanism by which volatile anesthetics produce their direct effects on vascular smooth muscle remains unknown. The authors previously reported that volatile anesthetics decrease both Ca2+ and K+ currents, however the role of Ca(2+)-activated K+ channels during the vasorelaxation by anesthetics has not been investigated. The purpose of this study was to determine whether blockade of the K+ channel alters the response to volatile anesthetics. Responses were studied in canine middle cerebral arteries and proximal and distal canine coronary arteries. Vascular rings (2-mm length) were suspended in tissue baths, and isometric tension was recorded. Rings were constricted with 40 mM KCl and prostaglandin F2 alpha (middle cerebral arteries only) and subsequently exposed to enflurane (3.25%), halothane (1.35%), and isoflurane (2.1%). Volatile anesthetics produced vasorelaxation with relative potency in order: enflurane > halothane > isoflurane. The procedure was repeated in the presence of the K+ channel blocker tetraethylammonium chloride (TEA, 20 mM). In all groups of vessels TEA alone elicited either no increase or only a transient increase in tension, however constrictions to both agonists were augmented in the presence of TEA. The presence of TEA significantly augmented anesthetic-induced vasorelaxation in small and large coronary vessels and in middle cerebral arteries. However, this effect was more pronounced in the cerebral as compared to coronary arteries. Constrictions produced in cerebral vessels by 15 microM prostaglandin F2 alpha were comparable with constrictions produced by 5 microM prostaglandin F2 alpha in the presence of TEA. The subsequent relaxant response of these vessels to enflurane was also comparable in the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of isoflurane on K+ and Ca2+ conductance in isolated smooth muscle cells of canine cerebral arteries.

Although isoflurane is a known cerebral vasodilator, the mechanism of isoflurane-induced vasodilation is not clear. The purpose of this study was to investigate the effects of 2.6% isoflurane (1.2 mM) on macroscopic calcium and potassium channel currents in voltage-clamped canine middle cerebral artery cells. Cells were dialyzed with K(+)-glutamate solution and superfused with Tyrode's solution for measurement of potassium current (n = 20). Stepwise depolarization from a holding potential of -60 mV to beyond -30 mV elicited an outward, slowly inactivating potassium current that was reduced 50% +/- 2% and 81% +/- 3% (mean +/- SEM) in the presence of 1 mM 4-aminopyridine and 30 mM tetraethylammonium, respectively. Calcium ionophore (A23187, 10 microM) increased the potassium current by 76% +/- 3%, suggesting calcium dependency. Isoflurane reduced the amplitude of the potassium current by 35% +/- 4%. Calcium current was measured in cells dialyzed with solution containing 130 mM Cs(+)-glutamate and superfused with solution containing 10 mM BaCl2 and 135 mM tetraethylammonium to pharmacologically isolate the calcium current (n = 13). Under these conditions, progressive depolarizing steps from -60 mV elicited an inward current that was maximally activated at +20 mV and essentially eliminated by 1 microM nifedipine. This current, resembling a long-lasting (L-type) Ca2+ channel current, was reduced 40% +/- 4% by isoflurane. The results of this study suggest that isoflurane acts directly at the vascular muscle membrane to suppress transmembrane calcium and potassium currents. The decrease in calcium current would cause vasodilation; however, the concomitant decrease in potassium current may partially antagonize the depressant effect of isoflurane mediated through calcium current reduction.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of guanylate cyclase-cGMP systems in halothane-induced vasodilation in canine cerebral arteries.

The cellular mechanisms through which halothane dilates blood vessels remain largely unknown. The present studies were designed to determine the effects of 0.59 and 0.9 mM halothane (equivalent to 2.0% and 3.0%, respectively) on tissue cyclic guanosine 3,5-monophosphate (cGMP) level and guanylate cyclase enzyme activity in canine middle cerebral arteries. Rings of cerebral arteries preconstricted with 5-hydroxytryptamine (0.2 microM) were exposed for 15 min to low or high concentrations of halothane or for 5 min to sodium nitroprusside (50 microM). The vessels were instantaneously frozen by immersing them in liquid N2; they then were homogenized, and the tissue cGMP levels were determined using radioimmunoassay. Halothane produced 2.23 +/- 0.44- and 4.47 +/- 0.87-fold increases in tissue cGMP levels over control at 0.59 and 0.9 mM, respectively. Sodium nitroprusside, a nitrovasodilator, also increased the tissue cGMP level 7.80 +/- 1.36-fold over the control value. To understand better the mechanisms of halothane-induced increase of tissue cGMP level, the effects of this anesthetic agent on guanylate cyclase enzyme activity were examined. Halothane, unlike sodium nitroprusside, did not modulate the activity of the soluble guanylate cyclase enzyme. However, halothane (1.0 mM), like atrial natriuretic factor (5 microM), stimulated the particulate guanylate cyclase enzyme activity. LY-83583 (6-anilino-5,8-quinolinedione, 10 microM), an agent that inhibits soluble guanylate cyclase activity, significantly reduced the response of the vessels to calcium ionophore (A23187, 0.4 microM), an endothelium-dependent vasodilator, without producing a significant effect on halothane-induced vasodilation. These results suggest that halothane-induced vasodilation of cerebral blood vessels is partly mediated by an increase in tissue cGMP levels.(ABSTRACT TRUNCATED AT 250 WORDS)