Canine Basilar Artery Research Articles

Specific inhibition of osmotic swelling-induced increase of L-type calcium channel current by herbimycin A in canine basilar artery

Specific inhibition of osmotic swelling-induced increase of L-type calcium channel current by herbimycin A in canine basilar artery

O–320 - Role of actin microfilament in stretch-induced contraction in canine basilar artery

O–320 - Role of actin microfilament in stretch-induced contraction in canine basilar artery

Effects of oxyhemoglobin on vasoconstriction in response to 5-hydroxytryptamine in isolated, perfused canine basilar arteries.

Oxyhemoglobin (OxyHb) is thought to be a critical trigger in the pathogenesis of cerebral vasospasm after aneurysmal subarachnoid hemorrhage. We investigated whether extraluminally applied OxyHb influenced vascular responses to intraluminally applied vasoactive agents in isolated, perfused, canine basilar arteries. The steel cannula insertion method was used to examine vascular responses to intraluminally applied 5-hydroxytryptamine (5-HT) receptor agonists, i.e., 5-HT, 5-carboxamidotryptamine (selective for 5-HT1 receptors), and alpha-methyl-5-hydroxytryptamine (selective for 5-HT2 receptors), potassium chloride, and acetylcholine, before and after extraluminal treatment with OxyHb. Extraluminal application of 2.5 x 10(-5) mol/L OxyHb immediately induced a transient elevation of the basal perfusion pressure, which gradually decreased and then stabilized at a level slightly higher than the control level. Each 5-HT agonist induced dose-dependent vasoconstriction. The potencies of the agonists were not very different, but the efficacies varied, i.e., alpha-methyl-5-hydroxytryptamine > 5-HT > 5-carboxamidotryptamine. Each response was strongly inhibited by ketanserin (a selective 5-HT2 receptor antagonist), indicating that each agonist induces vasoconstriction mediated by 5-HT2 receptors. The vasoconstriction in response to each 5-HT receptor agonist was consistently potentiated by treatment with OxyHb (2.5 x 10(-5) mol/L). 5-HT receptor agonist-induced constrictions after OxyHb treatment were much more markedly inhibited by ketanserin, compared with those before OxyHb treatment. Acetylcholine-induced constrictions were enhanced by OxyHb, but KCl-induced constrictions were significantly decreased by OxyHb. It is suggested that OxyHb enhancement of constrictions in response to 5-HT receptor agonists may be mediated by increased sensitivity of 5-HT2 receptors, in addition to actions in the endothelium, in canine basilar arteries. This potentiated vasoconstrictor mechanism may be partially implicated in cerebral vasospasm after subarachnoid hemorrhage.

Responses of Human Basilar and Other Isolated Arteries to Novel Nitric Oxide Donors

The adducts of nitric oxide (NO), diethylamine/NO (DEA/NO) and diethylenetriamine/NO (DETA/NO), are new NO donors that spontaneously release NO in aqueous solutions. These donors may have therapeutic advantages over sodium nitroprusside (SNP), which depends on metabolism to yield NO. This study was performed to define and compare the pharmacodynamic properties of these NO donors on isolated rings of human, canine, and porcine basilar arteries and further to compare canine and porcine common carotid arteries precontracted with KCl. The median effective concentration (EC50) and the basic effect of 100 microM were determined for each NO donor. On basilar arteries, DEA/NO was the most potent but the maximal dilatation produced by 100 microM did not persist for 60 min, whereas that of DETA/NO and SNP did. DETA/NO was more potent than SNP on all three species of basilar arteries but was the least potent on peripheral (carotid) arteries. Methylene blue in equimolar concentrations significantly inhibited the vasorelaxant effects of DEA/NO and DETA/NO, suggesting a common mechanism of action. Of the NO donors studied, the pharmacodynamic properties of DETA/NO seemed most relevant clinically as a cerebrovascular dilator in being more potent than SNP while producing sustained vasorelaxation.

Orz et al. report that a relatively pure solution of OxyHb contracts canine basilar arteries when applied extraluminally

Orz et al. report that a relatively pure solution of OxyHb contracts canine basilar arteries when applied extraluminally

Obligatory roles of protein kinase C and nitric oxide in the regulation of cerebral vascular tone: an implication of a pathogenesis of vasospasm after subarachnoid haemorrhage.

We previously showed that a canine basilar artery manifested tonic and potent, protein kinase C (PKC)-dependent contractions when nitric oxide (NO) was inhibited. We also reported a linear correlation between chronological changes in the angiographic severity of vasospasm, enhanced PKC, and attenuated guanosine, 13',15'-cyclic monophosphate (cGMP) activity in a canine subarachnoid haemorrhage model. The activity of cGMP is an indicator of NO-function. Based on this evidence, we have hypothesized that PKC and NO regulate cerebral vascular tone. We particularly focused on the role of NO in a negative feedback mechanism on PKC activity in the maintenance of vascular tone. To further confirm our hypothesis, we investigated the effect of PKC down-regulation on the tonic vascular contraction induced by NO-inhibition. Canine basilar artery was used in the experiment. Significant down-regulation of PKC activity in vascular smooth muscle cells was obtained by incubation with 10(-5) mole/L of phorbol 12-myristate 13-acetate (PMA) for 24 hours. The tonic and potent contraction induced by NO-inhibition was completely suppressed in the PKC down-regulated artery, even though the artery manifested a significant contraction in high-K+ solutions. These results indicate an obligatory role of PKC activity in tonic contraction when NO is inhibited, and support our previous data. Nitric oxide induces vascular relaxation by inhibiting PKC activity. Subarachnoid haemorrhage impairs this inhibition, resulting in PKC-dependent vascular contraction, such as vasospasm.

Interleukin-6 and development of vasospasm after subarachnoid haemorrhage.

The authors characterized the role of interleukins in the cerebrospinal fluid (CSF) in the development of vasospasm after subarachnoid haemorrhage (SAH), particularly interleukin-6 (IL-6). Concentrations of interleukin-1 beta (IL-1 beta), IL-6, and interleukin-8 (IL-8) were measured serially in CSF of 24 patients and in serum of 9 patients with SAH and correlated clinically. Additionally, the effects of the same cytokines on the cerebral arteries of dogs were analyzed on angiograms after intracisternal injection. Changes in levels of eicosanoids, angiogenic factors, and soluble cell adhesion molecules were investigated in the CSF of injected dogs. CSF concentrations of IL-6 and IL-8 were elevated significantly above control levels from the acute stage of SAH until the chronic stage. Patients with symptomatic vasospasm had significantly higher levels of IL-6 as well as IL-8 in CSF on days 5 and 7. Intracisternal injection of IL-6 induced long-lasting vasoconstriction in five out of eight dogs, while IL-8 did not. The diameter of canine basilar artery after IL-6 was reduced 29 +/- 5% from pretreatment diameter at 8 hours. Prostaglandins E2 and I2 were elevated in CSF for the first 4.5 hour of this IL-6-induced vasospasm. Neither angiogenic factors such as platelet-derived growth factor-AB and vascular endothelial growth factor nor soluble cell adhesion molecules were significantly elevated in CSF. IL-6, which increases to very high concentrations in CSF after SAH, may be important in inducing vasospasm, as IL-6 produced long-lasting vasoconstriction in the canine cerebral artery, which may be partly related to activation of the prostaglandin cascade.

Endothelium-dependent relaxation to hydrogen peroxide in canine basilar artery: a potential new cerebral dilator mechanism

In prostglandin F 2α(PGF 2α)-precontracted isolated canine basilar arterial rings, hydrogen peroxide (H 2O 2) produced endothelium-dependent relaxations at concentrations of from 4.4 × 10 −7–∼4.4 × 10 −5 M. Removal of extracellular Ca 2+ ([Ca 2+] 0) attenuated the relaxant effects of H 2O 2. Complete inhibition of H 2O 2 relaxant action was obtained after buffering intracellular Ca 2+ ([Ca 2+] i), in the endothelial cells, with 10 μM 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM). The H 2O 2-induced relaxations could be abolished completely by 1200 u/ml catalase and was suppressed significantly by 0.5 μM atropine, 150 μM N G-monomethyl-arginine (L-NMMA), 50 μM N G-nitro-L-arginine methyl ester (L-NAME), 1 μM Fe 2+, or 5 μM methylene blue. These inhibitory effects of L-NMMA, L-NAME, or atropine could be reversed partly by 50 μM L-arginine. The Fe 2+ inhibition of H 2O 2-stimulated relaxation was reduced significantly by either 1 mM deferoxamine (a Fe 2+ chelator) or 100 μM dimethyl sulfoxide (DMSO, a •OH scavenger). Such relaxant effects of H 2O 2 were enhanced, significantly, by an acetylcholinesterase antagonist, neostigmine. A variety of pharmacological antagonists (of diverse vasodilator agents) could not inhibit the relaxant action of H 2O 2. Our observations suggest that at suitable pathophysiological concentrations, H 2O 2 could induce release of an endothelium-derived relaxing factor (EDRF), probably nitric oxide (NO), from endothelial cells of the canine cerebral artery. The H 2O 2 relaxant effects are clearly Ca 2+-dependent, require formation of cyclic guanosine monophosphate (cGMP), and may be associated with release of endogenous acetylcholine (ACh).

Expression and function of recombinant endothelial nitric oxide synthase gene in canine basilar artery after experimental subarachnoid hemorrhage.

Gene transfer with recombinant viral vectors encoding vasodilator proteins may be useful in therapy of cerebral vasospasm after subarachnoid hemorrhage (SAH). Relaxations mediated by nitric oxide are impaired in cerebral arteries affected by SAH. The present study was designed to determine the effect of SAH on the efficiency of ex vivo adenovirus-mediated gene transfer to canine basilar arteries and to examine whether expression of recombinant endothelial nitric oxide synthase (eNOS) gene may have functional effects on vasomotor reactivity of spastic arteries affected by SAH. Replication-deficient recombinant adenovirus vectors encoding bovine eNOS (AdCMVeNOS) and Escherichia coli beta-galactosidase (AdCMVbeta-Gal) genes were used for ex vivo gene transfer. Rings of basilar arteries obtained from control dogs and dogs exposed to SAH were incubated with the vectors in minimum essential medium. Twenty-four hours after gene transfer, expression and function of the recombinant genes were evaluated by (1) histochemical or immunohistochemical staining, (2) beta-galactosidase protein measurement, and (3) isometric tension recording. Transduction with AdCMVbeta-Gal and AdCMVeNOS resulted in the expression of recombinant beta-galactosidase and eNOS proteins mostly in the vascular adventitia. The expression of beta-galactosidase protein was approximately 2-fold higher in SAH arteries than in normal arteries. Endothelium-dependent relaxations caused by bradykinin and substance P were suppressed in SAH arteries. The relaxations to bradykinin were significantly augmented in both normal and SAH arteries after AdCMVeNOS transduction but not after AdCMVbeta-Gal transduction. The relaxations to substance P were augmented by AdCMVeNOS transduction only in normal arteries. Bradykinin and substance P caused relaxations even in endothelium-denuded arteries, when the vessels were transduced with AdCMVeNOS. These endothelium-independent (adventitia-dependent) relaxations to bradykinin observed after AdCMVeNOS transduction were similar between normal and SAH arteries, whereas those to substance P were significantly reduced in SAH arteries compared with normal arteries. These results suggest that expression of recombinant proteins after adenovirus-mediated gene transfer may be enhanced in cerebral arteries affected by SAH and that successful eNOS gene transfer to spastic arteries can at least partly restore the impaired nitric oxide-mediated relaxations through local (adventitial) production of nitric oxide.

Levcromakalim decreases vascular tone, cytoplasmic Ca2+ and Ca2+ sensitivity in canine basilar artery.

The involvement of large conductance Ca(2+)-activated K+ channels (BK) and ATP-sensitive K+ (KATP) channels in the regulation of canine basilar arterial tone was estimated in the presence of the agonist and blockers of these channels, by simultaneously measuring the changes in intracellular Ca2+ concentration ([Ca2+]i) with the fura-2 microfluorimetric method. In the resting condition, levcromakalim reduced [Ca2+]i and vascular tone. Levcromakalim suppressed the serotonin-induced increases in [Ca2+]i and force of contraction, the maximum effects of which were much greater than those of nicardipine. The inhibitory effects of levcromakalim were blocked by glibenclamide but not by tetraethylammonium (TEA) or iberiotoxin (IbTX). In the presence of levcromakalim, the curve relating [Ca2+]i with force in the presence of serotonin at different extracellular Ca2+ concentration ([Ca2+]o) was shifted down- and right-ward compared with that in the absence of levcromakalim, suggesting that levcromakalim may reduce the Ca(2+)-sensitivity of the contractile proteins. Thus, levcromakalim may be a good candidate to suppress delayed cerebral vasospasm after subarachnoid hemorrhage.

Impairment in biochemical level of arterial dilative capability of a cyclic nucleotides-dependent pathway by induced vasospasm in the canine basilar artery.

The authors investigated the changes and the potential of cyclic nucleotide-dependent signal transduction, which induces smooth muscle relaxation, in the basilar artery with severe vasospasm in dogs with double experimental subarachnoid hemorrhage (SAH) to explore at which biochemical level the arterial dilative capability was impaired. The amount of cyclic adenosine and guanosine monophosphates (cAMP and cGMP) decreased significantly in the basilar artery after SAH. The activities of adenylate and guanylate cyclases also were decreased significantly in the smooth muscle cells of the basilar artery 4 days after SAH. In addition to the failure of the pathways to produce cyclic nucleotides, the activities of cAMP- and cGMP-dependent protein kinases, which are representative actual enzymes that amplify the signal for vascular dilation, also significantly decreased together with the almost total loss of activation by cyclic nucleotides in the same basilar artery after SAH. It was revealed that the system for smooth muscle relaxation was impaired severely in the cerebral arteries with severe vasospasm after SAH, on the biochemical basis of significantly less vasodilative capability and in several of the steps to produce the cyclic nucleotides of intracellular signal transduction.

Inducible cyclooxygenase expression in canine basilar artery after experimental subarachnoid hemorrhage.

Inducible cyclooxygenase (COX-2) has been found to play a pathological role in cerebral insult. We investigated the expression of COX-2 in the basilar artery after experimental subarachnoid hemorrhage (SAH). In a canine "two-hemorrhage" model of SAH, the basilar arteries were obtained on day 2 after a cisternal injection of autologous blood or on days 4, 6, 7, or 9 after the second injection. Basilar arteries also were obtained 12 hours after intracisternal injection a cytokine: interleukin (IL)-1 beta (0.03 microgram), IL-6 (3 micrograms), or IL-8 (10 micrograms). Western blotting with a polyclonal anti-COX-2 antibody was performed in these arteries. COX-2 protein was not demonstrated in the basilar artery in control animals without SAH. However, it was expressed in the basilar artery on days 2, 4, 6, and 7 after blood injection but not on day 9. Intracisternal injection of IL-1 beta, IL-6, or IL-8 also induced COX-2 in the basilar artery. COX-2 expression was detected in basilar arterial tissue in both acute and chronic stages after SAH. Elevation of inflammatory cytokines after SAH may be involved in the induction of COX-2, which may produce sufficient quantities of eicosanoids to affect hemodynamics after SAH.

Subarachnoid hemorrhage and the role of potassium channels in relaxations of canine basilar artery to nitrovasodilators.

This study was designed to determine the effect of subarachnoid hemorrhage (SAH) on potassium (K+) channels involved in relaxations of cerebral arteries to nitrovasodilators. The effects of K+ channel inhibitors on relaxations to 3-morpholinosydnonimine (SIN-1) and sodium nitroprusside (SNP) were studied in rings of basilar arteries obtained from untreated dogs and dogs exposed to SAH. The levels of cyclic GMP were measured by radioimmunoassay. In rings without endothelium, concentration-dependent relaxations to SIN-1 (10(-9)-10(-4) mol/L) and SNP (10(-9)-10(-4) mol/L) were not affected by SAH, whereas increase in cyclic GMP production stimulated by SIN-1 (10(-6) mol/L) was significantly suppressed after SAH. The relaxations to SIN-1 and SNP were reduced by charybdotoxin (CTX: 10(-7) mol/L), a selective Ca(2+)-activated K+ channel inhibitor, in both normal and SAH arteries; however, the reduction of relaxations by CTX was significantly greater in SAH arteries. By contrast, the relaxations to these nitrovasodilators were not affected by glyburide (10(-5) mol/L), an ATP-sensitive K+ channel inhibitor, in both normal and SAH arteries. These findings suggest that in cerebral arteries exposed to SAH, CA(2+)-activated K+ channels may play a compensatory role in mediation of relaxations to nitric oxide. This may help to explain mechanisms of relaxations to nitrovasodilators in arteries with impaired production of cyclic GMP.

Change in Ca2+ sensitivity of cerebrovascular smooth muscle in experimental chronic cerebral vasospasm.

The mechanism of chronic cerebral vasospasm was investigated by examining the relation between cytosolic Ca2+ level ([Ca2+]i) and muscle contraction in vascular smooth muscle after experimental vasospasm produced by the two-hemorrhage method in the canine basilar artery. [Ca2+]i and tension were recorded simultaneously with a fluorimeter using the arterial strips with a Ca2+ indicator, fura-2. High K+ concentration (72.4 mM) and U-46619 (thromboxane A2 analogue, 10(-8) M) were used as stimulants. The increase in contractile tension per [Ca2+]i per unit cross-sectional area was calculated, and compared for the vasospasm and the control groups. The increase in [Ca2+]i in the vasospasm group was smaller than that in the control group, so the increase in tension per [Ca2+]i per unit sectional area was larger in the vasospasm group. This value was much larger for the contraction induced by U-46619 than that induced by high K+ concentration in both groups. These results suggest that there is an increase in the basal Ca2+ sensitivity of the contractile elements in vasospastic vessels. In addition, the U-46619-induced increase in Ca2+ sensitivity seemed to be augmented in the vasospasm group.

Endothelium is required for 12-hydroperoxyeicosatetraenoic acid-induced vasoconstriction

The pharmacological effects of 12-hydroperoxyeicosatetraenoic acid (12-HPETE) were examined using isolated canine basilar artery segments and isometric tension recording. 12-HPETE produced transient contraction of the artery segment while arachidonic acid or 12-hydroxyeicosatetraenoic acid (12-HETE) had a much lower potency. 12-HPETE-induced contraction which showed a requirement of a functional endothelium and a rapid insensitivity to re-administered 12-HPETE, was completely inhibited by the potassium channel blocker, glibenclamide. Other hydroperoxides did cross-desensitize the 12-HPETE-induced contraction, however, arachidonic acid or 12-HETE did not affect markedly. Here, we present that 12-HPETE is involved in the regulation of vascular tension via its effects on the endothelium.

P-312 - Levcromakalim decreases cytoplasmic Ca2+ and vascular tone in canine basilar artery of SAH model

P-312 - Levcromakalim decreases cytoplasmic Ca2+ and vascular tone in canine basilar artery of SAH model

Vasodilator effects on canine basilar artery induced by intracisternal interleukin-1 beta.

The effect of interleukin-1 beta (IL-1 beta) on a cerebral artery was investigated in anesthetized dogs. Intracisternal administration of IL-1 beta (0.03 and 0.3 micrograms) dilated the canine basilar artery in a dose-dependent manner, without affecting systemic blood pressure or heart rate. The increase in diameter induced by 0.3 micrograms of IL-1 beta was 28.4% +/- 13.4% of control at 2 hours and was inhibited by 30 micrograms of the IL-1 beta receptor antagonist, zinc protoporphyrin (4.5% +/- 13.5%, P < 0.05). Interleukin-1 beta did not affect the concentration of nitric oxide metabolites in CSF. However, there was an increase in the concentration of eicosanoids in CSF, and the elevation of 6-keto-PGF1 alpha paralleled the vasodilation. Pretreatment with 30 micrograms of the selective inducible cyclooxygenase (COX-2) inhibitor NS-398 also inhibited the IL-1 beta-induced vasodilation significantly (5.9% +/- 9.4% at 2 hours, P < 0.01). Western blot analysis revealed the expression of a 68-kD COX-2-like protein in basilar artery extracts. These findings suggest that the IL-1 beta-induced vasodilator effect is linked to the prostaglandin cascade, predominantly to prostaglandin I2, by induction of COX-2, but not to the stimulation of nitric oxide metabolism.

Generation of the catalytic fragment of protein kinase C alpha in spastic canine basilar artery.

In previous studies of topical application of calphostin C, a specific inhibitor of the regulatory domain of protein kinase C (PKC), and calpeptin, a selective inhibitor of calpain, to spastic canine basilar artery (BA) researchers have suggested that the catalytic fragment of PKC (known as PKM) is probably formed by a limited proteolysis of continuously activated mu-calpain, but there has been no direct evidence for PKM formation in vasospasm. The present immunoblot study with anti-PKCalpha antibody shows a significant decrease in cytosolic 80-kD PKCalpha and a concomitantly significant increase in membrane PKCalpha in the spastic canine BA. In addition, an immunoblot study in which cleavage site-directed antibodies were used demonstrated a significant increase in immunoreactive 45-kD PKM. The changes in membrane PKCalpha and PKM were enhanced with the lapse of time after subarachnoid hemorrhage. The cleavage site-directed antibodies distinguish the proteolyzed from the unproteolyzed forms of PKC for in situ analyses of enzyme regulation mediated by proteolysis. The data indicate that PKCalpha in spastic canine BA is translocated to the cell membrane, where PKCalpha is rapidly cleaved into PKM as a result of proteolysis of the isozyme by mu-calpain but not by m-calpain. The authors hypothesize that mu-calpain is continuously activated in spastic canine BA and produces PKM by limited proteolysis of PKCalpha.

Generation of the catalytic fragment of protein kinase C alpha in vasospastic canine basilar artery

In previous studies of topical application of calphostin C, a specific inhibitor of the regulatory domain of protein kinase C (PKC), and calpeptin, a selective inhibitor of calpain, to spastic canine basilar artery (BA) researchers have suggested that the catalytic fragment of PKC (known as PKM) is probably formed by a limited proteolysis of continuously activated μ-calpain, but there has been no direct evidence for PKM formation in vasospasm. The present immunoblot study with anti-PKC-alpha antibody shows a significant decrease in cytosolic 80-kD PKC-alpha and a concomitantly significant increase in membrane PKC-alpha in the spastic canine BA. In addition, an immunoblot study in which cleavage site-directed antibodies were used demonstrated a significant increase in immunoreactive 45-kD PKM. The changes in membrane PKC-alpha and PKM were enhanced with the lapse of time after subarachnoid hemorrhage. The cleavage site-directed antibodies distinguish the proteolyzed from the unproteolyzed forms of PKC for in situ analyses of enzyme regulation mediated by proteolysis. The data indicate that PKC-alpha in spastic canine BA is translocated to the cell membrane, where PKC-alpha is rapidly cleaved into PKM as a result of proteolysis of the isozyme by μ-calpain but not by m-calpain. The authors hypothesize that μ-calpain is continuously activated in spastic canine BA and produces PKM by limited proteolysis of PKC-alpha.

Dysfunction of nitric oxide induces protein kinase C activation resulting in vasospasm after subarachnoid hemorrhage

We hypothesize that the interaction between protein kinase C (PKC) and nitric oxide (NO) plays a role in the modulation of cerebral vascular tone, and the disturbance of this interaction following subarachnoid hemorrhage (SAH) results in vasospasm. To prove this hypothesJs with direct evidence, PKC activities of smooth muscle cells of canine basilar arteries in the control and in the SAH groups were measured by an enzyme immunoassay method. Nw-nitro-L arginine (L-NA), an inhibitor of NO production, enhanced PKC activity. This enhancement was inhibited neither by 8-bromo-guanosine 3’5’-cyclic monophosphate (8-bronio-cGMP) nor SIN-7, a NO releasing agent. PKC activity in the SAH was significantly higher than in the control; however, no furtherenhancement was produced with L-NA. In the SAH, PKC activity was not inhibited either by 8-bromo~cGMPor SIN-7. We conclude that NO maintains an appropriate vascular tone through inactivation of PKC, and that this effect is disturbed following SAH, resulting in PKC-dependent vascular contraction, such as vasospasm. On the other hand, once PKC has been activated, NO precursors do not inhibit PKC. These facts indicate NO inactivates PKC through the inhibition of phosphatidylinositol breakdown. [Neural Res 1997; 19: 558-562]