Cannula Inserting Method Research Articles

Antagonistic Interaction Between BIIE 0246, a Neuropeptide Y Y2-Receptor Antagonist, and ωy-Conotoxin GVIA, a Ca2+ Channel Antagonist, in Presynaptic Transmitter Releases in Dog Splenic Arteries

Isolated dog splenic arteries were perfused with Krebs-Henseleit solution at 37°C, using the cannula inserting method. Periarterial nerve electrical stimulation (10-V amplitude; 1-ms duration; 30-s trains of pulses; 1, 4 and 10 Hz) readily caused double peaked vasoconstrictions, i.e., 1st peaked response was mostly inhibited by α, β-methylene ATP and the 2nd one, by prazosin. These responses were consistently inhibited by ω-conotoxin GVIA (ω-CTX), whereas they were facilitated by BIIE 0246, a neuropeptide Y (NPY) Y2-receptor antagonist. The ω-CTX-induced blocking effects of transmitter release were significantly antagonized by BIIE 0246. It is possible that the NPY Y2 receptor activity may partially be linked to presynaptic Ca2+ channels.

Existence of Functional α1A- and α1D- but No α1B-Adrenoceptor Subtypes in Rat Common Carotid Arteries

Using the cannula inserting method, vasoconstrictor responses to alpha1-adrenoceptor agonists (noradrenaline [NA], phenylephrine [PE] and methoxamine [ME]) and effects of alpha1-adrenoceptor antagonists (WB4101, chloroethylclonidine [CEC] and BMY7378) were investigated in isolated and perfused rat common carotid arteries. The rank order of agonist potency and efficacy was NA = PE > ME. Either WB4101 or BYM7378 inhibited NA- and PE-induced constrictions in a dose-related manner. CEC did not inhibit the NA- and PE-induced responses. The ME-induced responses were also significantly blocked by either WB4101 or BMY7378. From these results, it is concluded that there are functional alpha1A- and alpha1D-adrenoceptor subtypes in rat common carotid arteries, but no functional alpha1B subtype.

Vascular responses to beta-adrenoceptor subtype-selective agonists with and without endothelium in rat common carotid arteries.

1. Using the cannula inserting method, vasodilator responses to beta-adrenoceptor agonists (isoprenaline, denopamine and procaterol) were investigated in isolated and perfused rat common carotid arteries. 2. Each beta-adrenoceptor agonist induced a vasodilation in preparations preconstricted by phenylephrine in a dose-related manner. The potencies were in the order of isoprenaline > procaterol >> denopamine. 3. Denopamine-induced dilations were significantly inhibited by 1 nmol betaxolol (a selective beta1-adrenoceptor antagonist), but it was not influenced by 1 nmol ICI 118,551 (a selective beta2-adrenoceptor antagonist). On the other hand, procaterol-induced vasodilations were significantly inhibited by 1 nmol ICI 118,551 but not modified by 10 nmol betaxolol. 4. ACh-induced vasodilations disappeared after intraluminal saponin injection to remove endothelium, but procaterol- and denopamine-induced dilations were not modified by removal of the endothelium. 5. Pretreatment with L-NG-nitroarginine methyl ester (L-NAME) readily inhibited ACh-induced vasodilations. However, neither procaterol- or denopamine-induced vasodilation was modified by L-NAME treatment. 6. From these results, it is concluded that in the rat common carotid arteries (1) there are abundant beta2- and a few beta1-adrenoceptors, and (2) there is no participation of the endothelium-dependent mechanism in beta-adrenoceptor mediated vasodilations.

Effect of temperature on responses of dog isolated lingual and mesenteric arteries to vasoactive substances.

1. The effects of temperature on submaximal vasoconstriction to an intraluminal administration of noradrenaline (NA), phenylephrine, tyramine and KCl were investigated in canine isolated and perfused lingual and mesenteric arteries, using the cannula-inserting method. 2. In lingual arteries, cooling (from 37 to 27 degrees C) caused significant depression of vasoconstriction to the four vasoactive substances used. Rewarming (to 37 degrees C) induced a significant augmentation of constriction by NA, phenylephrine and KCl, but not tyramine. 3. In mesenteric arteries, cooling depressed tyramine- and KCl-induced constrictions, but had no effect on NA- and phenylephrine-induced vasoconstriction. Only in the case of KCl-induced constrictions did rewarming induce a potentiation of the vasoconstrictor response. 4. We conclude that: (i) cooling induces a depression of voltage-dependent Ca2+ channels and rewarming may induce a potentiation of Ca2+ channels in both arteries; (ii) alpha1-adrenoceptor-operated Ca2+ channels are depressed by cooling in lingual arteries but not in mesenteric arteries; and (iii) cooling may induce an attenuation of the re-uptake function in sympathetic nerve terminals in both arteries and this attenuation may be not rapidly restored by acute rewarming.

ROLE OF ENDOTHELIUM AND VASOCONSTRICTOR PROSTANOIDS IN NOREPINEPHRINE-INDUCED VASOCONSTRICTION IN ISOLATED RAT COMMON CAROTID ARTERIES

We investigated the role of endothelium and vasoconstrictor prostanoids in the norepinephrine (NE)-induced vasoconstriction of isolated rat common carotid arteries (CCAs). Isolated CCAs were cannulated with stainless steel cannulae by the cannula inserting method. NE was administered intra- or extraluminally by a single microinjection. For denudation of endothelium, the intimal surface was gently rubbed with a cotton pellet. NE produced dose-related vasoconstricting responses in isolated arteries with intact endothelium. These responses were attenuated by pretreatment with denudation, OKY046, a thromboxane A2 synthesis inhibitor or indomethacin. The residual responses after denudation were further blocked by OKY046 or indomethacin. All NE-induced responses were blocked by a single injection of prazosin. There were no significant differences in responses between intra- and extraluminal administration of NE. These results show that most vasoconstriction induced by NE via α1-adrenoceptors in CCAs is dependent on vasoconstrictor prostanoids distributed both in endothelium and vascular smooth muscle.

Effect of ketanserin on 5-hydroxytryptamine-induced constriction in isolated, perfused canine basilar arteries exposed to blood

The blocking effect of ketanserin, a 5-hydroxytryptamine (5-HT)2 receptor antagonist, was examined on responses to intra-luminal 5-HT or potassium chloride (KCl) in isolated and perfused canine basilar arteries before and after extraluminal whole blood by means of the stainless-steel cannula inserting method. Four hours after the application of blood, the constriction to 5-HT was significantly enhanced, while that to KCl was significantly attenuated. The absolute value in the decrease of perfusion pressure for the 5-HT-induced constriction sensitive to ketanserin was much greater after the blood application than that before the application. It is suggested that the augmentation of cerebrovascular responses to 5-HT in the early stage immediately after subarachnoid haemorrhage (SAH) might be mediated by 5-HT2 receptors at least in part. This early constrictor mechanism may be implicated in the aetiology of the following late stage of cerebral vasospasm after SAH.

Vasoconstrictor mechanism of neuropeptides augmented after endothelial removal in isolated, perfused canine basilar arteries

In order to investigate the functional role of endothelium and vasoeffector mechanism in cerebrovascular responses to neuropeptides, the stainless steel cannula inserting method was applied to examine the responses to intraluminally-applied bradykinin, substance P and vasopressin in isolated and perfused canine basilar arteries. In control vessels with intact endothelium, each neuropeptide induced a monophasic dilation at lower doses, and a biphasic response, i.e., an initial dilation followed by a secondary constriction, at higher doses. The dilation was significantly reduced and the constriction was significantly enhanced, while the dilation to papaverine was not modified by endothelial removal with intraluminal saponin. The same tendency was observed in the responses after extraluminal treatment with oxyhaemoglobin. The monophasic constrictions to prostaglandin F2 alpha and potassium chloride were significantly potentiated by the endothelial removal. The augmented constrictions to the neuropeptides were significantly diminished by indomethacin (a cyclooxygenase inhibitor), OKY-046 (a thromboxane synthetase inhibitor) and nimodipine (a dihydropyridine calcium antagonist), but not by AA-861 (a lipoxygenase inhibitor). These results suggest that the neuropeptide causes an endothelium-dependent dilation and a constriction of smooth muscles, and that the enhanced constriction might be relevant in part with thromboxane A2, linked with calcium influx into smooth muscle cells in cerebral arteries.

Mechanism of Acetylcholine-Induced Constriction Enhanced by Endothelial Removal in Isolated, Perfused Canine Basilar Arteries

We examined the responses to intraluminally applied acetylcholine (ACh) in isolated and perfused canine basilar arteries by the stainless-steel cannula-inserting method. In control vessels with intact endothelium, ACh produced a slight vasodilatation followed by a long-lasting vasoconstriction in the absence of induced tone. Propranolol, a beta-adrenoceptor blocker, had no effect on the response to ACh. After endothelial removal with intraluminal saponin, the constrictor component of the response to ACh was significantly enhanced, and the constrictions to prostaglandin F2 alpha (PGF2 alpha) and potassium chloride (KCl) were significantly potentiated. After exposure to extraluminal oxyhemoglobin, the ACh-induced constriction was significantly augmented, whereas the dilator component of the response to calcium ionophore A23187 was significantly attenuated. The enhanced constriction to ACh after endothelial removal was significantly blocked by OKY-046 (a thromboxane synthetase inhibitor) and nimodipine (a dihydropyridine calcium antagonist), but was slightly though not significantly suppressed by AA-861 (a lipoxygenase inhibitor). The response to PGF2 alpha was not altered by OKY-046 or AA-861. These results suggest that the potentiation of ACh-induced constriction after endothelial removal may be mediated in part by thromboxane A2 (TXA2), linked with extracellular calcium entry in smooth muscle cells (SMC). This mechanism might be involved in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH).

A new cannula-inserting method for measuring vascular responsiveness separate intraluminal and extraluminal perfusion of canine basilar artery

We modified the cannula-inserting method to examine the difference in response of vessels to intraluminal or extraluminal application of vasoactive substances. A polythylene roof was designed to cover the isolated canine basilar artery so that an extraluminal superfusion stream could pass over an artery that simulataneously received an intraluminal perfusion. An extraluminal administration of KCl produced a monophasic transient vasoconstriction, but intraluminal KCl produced relatively small tonic constriction even at high concentrations. Serotonin-induced vasoconstrictions were significantly larger with intraluminal administration than with extraluminal. Prostaglandin F 2α-induced vasoconstriction exhibited no significant difference between the administration routes. Endothelin caused a long-lasting constriction, which was slightly larger in extra- than intraluminal administration. Our modified cannula-inserting technique provides a new method to examine the different effects produced by intraluminal and extraluminal application of drugs.

A new perfusion technique for vascular vessels

A new perfusion technique for isolated vascular preparations is described. The cannula inserting method developed by Hongo and Chiba (J. Pharmacol. Methods 9, 83, 1983) and modified by Tsuji and Chiba (Japan. J. Pharmacol. 34, 95, 1984) is useful for observing vascular reactivity in perfused, isolated vessels. In this method, 1) applied substances act only from the intraluminal side; 2) vascular responses to substances, not only constriction but also dilatation in non-treated preparations, are readily obtainable by a single injection of active substances; and 3) the vascular reactivity is reproducible over 7-8 hr at 37 degrees C. Our modified cannula inserting methods are also described.

Vascular Responsiveness of Rabbit Common Carotid, Renal and Femoral Arteries to α-Adrenoceptor Agonists

The stainless steel cannula inserting method was used to observe vascular effects of alpha-adrenoceptor agonists, phenylephrine (PE), methoxamine (ME), clonidine (CL) and xylazine (XY), on the isolated, perfused rabbit common carotid, renal and femoral arteries. PE and ME induced a dose-dependent vasoconstriction that was readily suppressed by treatment with bunazosin, an alpha 1-adrenoceptor blocker. CL induced a constriction only in common carotid arteries, and this was readily suppressed by bunazosin. In preparations preconstricted by phenylephrine, CL and XY dose-dependently induced vasodilations in the 3 types of arteries, and these vasodilations were not modified by a potent alpha 2-adrenoceptor antagonist, midaglizole. In preparations preconstricted by prostaglandin F2 alpha, CL and and XY did not produce any significant vasodilation, but CL induced a vasoconstriction in common carotid arteries that was completely blocked by bunazosin. Thus, it is concluded that: 1) alpha 1-adrenoceptors are functionally predominant in rabbit peripheral arteries, 2) the alpha 2-adrenoceptor agonist-induced vasodilation may be due to an antagonistic action towards alpha 1-mediated constrictions, and 3) clonidine has alpha 1-adrenoceptor stimulating properties in rabbit common carotid artery but not in renal and femoral arteries.

Biphasic Response to Substance P in Canine Basilar Arteries

The responses to intraluminally applied substance P (SP) were examined in isolated and perfused canine basilar arteries using the stainless-steel cannula inserting method. In control vessels with intact endothelium, this peptide induced a monophasic dilation at lower doses, and a biphasic response, i.e., an initial dilation followed by a secondary constriction at higher doses. After extraluminal treatment with oxyhemoglobin, the dilation was attenuated and the constriction was augmented. After endothelial removal with intraluminal saponin, the dilation was reduced and the constriction was enhanced significantly. This potentiated constriction was significantly depressed by indomethacin (a cyclooxygenase inhibitor), OKY-046 (a thromboxane synthetase inhibitor), and nimodipine (a calcium antagonist), but not by AA-861 (a lipoxygenase inhibitor). These results suggest that SP has two distinct effects (an endothelium-dependent dilation and a direct constriction) and that the potentiated constriction in the absence of endothelium may be related to the action of thromboxane A2, linked with calcium influx into the smooth muscle cells of cerebral arteries. This mechanism may be implicated in cerebral vasospasm after subarachnoid hemorrhage.

Histamine-induced vasodilations mediated by H1- and H2-receptors in isolated rat common carotid arteries

Using the cannula inserting method, the vasodilatory effects of histamine were analysed employing selective histamine H1- and H2-receptor agonists and antagonists in isolated, perfused rat common carotid arterial preparations which were preconstricted by a continuous infusion of phenylephrine with propranolol. Histamine, 2-pyridylethylamine (2-PEA) (a selective H1-agonist) and dimaprit (a selective H2-agonist) produced a vasodilation in a dose-related manner. The order of potency was histamine greater than dimaprit greater than 2-PEA. Histamine-induced dilations were significantly inhibited by either diphenhydramine (a selective H1-antagonist) or cimetidine (a selective H2-antagonist). 2-PEA-induced dilations were significantly inhibited by diphenhydramine but not by cimetidine. Dimaprit-induced dilations were significantly blocked by cimetidine but not by diphenhydramine. ACh-, histamine-, 2-PEA- and dimaprit-induced dilations were significantly suppressed by removal of the endothelium. From these results, it is concluded that (1) isolated rat common carotid arteries have both H1- and H2-receptors, (2) there are few vasoconstrictory H1-receptors, (3) both H1- and H2-receptors mediate only vasodilation but not vasoconstriction, and (4) EDRF from the endothelium might participate in histamine-induced vasodilation via not only H1- but also H2-receptors.

Pharmacological analysis of vasoconstrictor responses of isolated and perfused human umbilical arteries

Using the cannula inserting method, we studied vascular responses of isolated human umbilical arteries to several vasoactive substances. ACh did not produce a vasodilation in non-constricted preparations but induced only a vasoconstriction. Histamine and 5HT produced strong vasoconstrictions in a dose-dependent manner. Epinephrine and norepinephrine in large doses induced only a slight vasoconstriction. The ACh-induced vasoconstriction was markedly suppressed by atropine and slightly, but significantly, suppressed by methylsergide. The vascular responses to ACh were not influenced by removal of the endothelium by an intraluminal bolus injection of saponin. These results suggest that the endothelium has no muscarinic receptors in the umbilical arteries, although cholinergic vasoconstrictor mechanisms may be partially involved in the regulation of umbilical circulation, and that human umbilical arteries exhibit different pharmacological responses from those of vessels of other organs.

Characteristics of Vascular Responses to Endothelin-1 of Simian Digital Arteries that Distribute Their Branches to the Skin

Using the cannula-inserting method, vascular responses to endothelin-1 were investigated in the isolated and perfused simian digital artery. Endothelin-1 produced a strong and sustained vasoconstriction with EC50 of 5.15 +/- 1.24 ng. The endothelin-1-induced vasoconstriction was significantly inhibited in the Ca(++)-removed perfusate. It was partly attenuated by diltiazem, a potent Ca++ entry blocker, but not affected by alpha-adrenoceptor antagonist phentolamine, indicating that endothelin-1 causes vasoconstriction by either the extracellular Ca++ entry or intracellular movement of Ca++ from the cytosolic storage site. From these results, it is suspected that ET-1 is partially related to skin circulatory disturbances such as Raynaud's phenomenon.

Evidence for the existence of postsynaptic beta-1 and beta-2 adrenoceptors in isolated simian facial veins

With the use of a steel cannula inserting method, the actions of the beta-adrenoceptor agonists, noradrenaline (NA, a mixed agonist), isoprenaline (a mixed agonist), dobutamine (a selective beta-1 agonist), salbutamol, and procaterol (selective beta-2 agonists), were investigated on isolated and perfused simian facial veins. Each beta-agonist usually induced a vasodilation in a dose-related manner in non-preconstricted vessel preparations. The rank order of potency was isoprenaline much greater than NA greater than dobutamine greater than salbutamol greater than procaterol. NA- and isoprenaline-induced vasodilations were inhibited by either metoprolol (a selective beta-1 adrenoceptor antagonist) or ICI 118,551 (a selective beta-2 antagonist). After beta-1 blockade, NA produced a vasoconstriction which was readily blocked by bunazosin (an alpha-1 antagonist). Dobutamine-induced vasodilations were strongly suppressed by metoprolol and slightly blocked by ICI 118,551. Salbutamol-induced vasodilations were blocked by metoprolol, while ICI 118,551 more markedly inhibited these dilations. From these results, it was concluded that there are abundant beta-adrenoceptors and predominantly beta-1 adrenoceptors in isolated simian facial veins.

Muscarinic Receptor Subtype in Isolated Dog and Monkey Facial Vein

Using the cannula inserting method, we investigated vascular responses to ACh and McN-A-343 (a M1-agonist) in isolated and perfused canine and simian facial veins in non-preconstricted conditions. ACh usually induced only a vasoconstriction, but McN-A-343 did not induce any significant vasoconstriction. It is concluded that canine and simian facial veins contain very few receptors of the muscarinic M1-subtype; and according to previous studies, these vessels have abundant M3-receptors.

Muscarinic receptor subtype in isolated dog and monkey facial vein.

Using the cannula inserting method, we investigated vascular responses to ACh and McN-A-343 (a M1-agonist) in isolated and perfused canine and simian facial veins in non-preconstricted conditions. ACh usually induced only a vasoconstriction, but McN-A-343 did not induce any significant vasoconstriction. It is concluded that canine and simian facial veins contain very few receptors of the muscarinic M1-subtype; and according to previous studies, these vessels have abundant M3-receptors.

Perfusion-time dependent enhancements of guanabenz- and KCl-induced vasoconstrictions in isolated and perfused dog pulmonary veins

The responses of the isolated canine pulmonary vein to guanabenz and to potassium chloride (KCl) were examined by means of the cannula-inserting method. The vessel was perfused by Krebs-Ringer bicarbonate solution at a constant flow rate at 37 degrees C. In the early stage (within 2-3 hours of the perfusion period), guanabenz induced a dose-dependent vasoconstriction, whereas high doses of KCl caused, at most, a slight vasoconstriction. At the late stage (within 8-11 hours of the perfusion period), the dose-response curve for guanabenz was shifted to the left and the maximum response became approximately 3-fold larger than that in the early stage. Vasoconstriction in response to KCl was observed in 8 out of 11 preparations (73%) in the late stage. In both early and late stages, guanabenz-induced responses were similarly antagonized by DG-5128 (a selective alpha 2-adrenoceptor antagonist) but not by bunazosin (a selective alpha 1-adrenoceptor antagonist). Diltiazem caused an inhibition of guanabenz-induced constrictions, particularly in the late stage. KCl-induced vasoconstriction was not affected by bunazosin or DG-5128, but was markedly suppressed by diltiazem. In preparations in which the endothelium was removed by intraluminal treatment with saponin, the changes in guanabenz- and KCl-induced responses, which were dependent on the perfusion time, were not modified in either stage. Our results suggest that alpha 2-adrenoceptor-mediated vasoconstriction is enhanced in a perfusion-time-dependent manner in the isolated and perfused canine pulmonary vein. These enhanced responses mediated by latent alpha 2-adrenoceptors are dependent on the influx of extracellular calcium ions.(ABSTRACT TRUNCATED AT 250 WORDS)

Dominant Vasodilator Action of Norepinephrine in Isolated, Non-Preconstricted Simian Facial Veins

Using the cannula inserting method, we investigated the vascular responses to norepinephrine (NE), phenylephrine and isoprenaline (Isp) in isolated, perfused simian facial veins. NE usually induced only a vasodilation in non-preconstricted veins. Phenylephrine consistently induced only a slight vasoconstriction, and Isp produced only a vasodilation. NE-induced vasodilations were reversed to vasoconstrictions after treatment with a relatively larger dose of propranolol. It is concluded that simian facial veins have a spontaneous intrinsic tone and dominant abundant beta-adrenoceptors but sparse alpha1-adrenoceptors.