Contraction followed by relaxation in response to hypoxia in the sheep isolated middle cerebral artery

In rings of sheep middle cerebral artery, precontracted with 5-hydroxytryptamine (5-HT) 10 microM, hypoxia (pO2 = 10 mm Hg) caused contraction and re-oxygenation caused a transient further relaxation. The hypoxic contraction was abolished and the re-oxygenation relaxation was reduced by removal of the endothelium. BW755C 20 microM reduced the hypoxic contraction and re-oxygenation relaxation. Haemolysate 1 microliter/ml and methylene blue 10 microM reduced the hypoxic contraction and re-oxygenation relaxation and enhanced the hypoxic relaxation phase. It is concluded that the endothelium of isolated rings of sheep middle cerebral artery releases vasoconstrictor substances during hypoxia and releases vasodilator substances during re-oxygenation.     

Histamine increases vascular tone and intracellular calcium level using both intracellular and extracellular calcium in porcine coronary arteries

Effects of histamine on the tone and intracellular calcium level (Ca2+i) in porcine coronary arteries were simultaneously investigated by use of the fura-2 microscopic fluorometric method. Histamine (10(-6)-10(-4) M) induced concentration-dependent increases in tone and Ca2+i, but these responses were not sustained. Histamine induced a larger contraction than did KCl with a similar increase in Ca2+i. Depletion of the caffeine-sensitive Ca2+ store with ryanodine (3 x 10(-5) M) and repetitive applications of caffeine (2.5 x 10(-2) M) scarcely affected contractile and Ca2+i responses to histamine. In Ca2(+)-free medium or in the presence of verapamil (10(-6) M), histamine produced a briefer increase in Ca2+i and a smaller contraction than in normal medium. When histamine or caffeine was repetitively applied in Ca2(+)-free medium, the first application produced an increase in Ca2+i but the second application produced no increase. Although caffeine increased Ca2+i after repetitive histamine applications, histamine failed to increase Ca2+i after repetitive caffeine applications in Ca2(+)-free medium. These results indicate that vascular contraction induced by histamine may involve the following mechanisms: an increase in Ca2+ influx through Ca2+ channels, release of Ca2+ from the intracellular Ca2+ store which has an interaction with the caffeine-sensitive Ca2+ store, and sensitization of contractile proteins to Ca2+.     

Pharmacological regulators of intracellular calcium release channels

Intracellular Ca(2+) release channels, such as inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs), facilitate the release of Ca(2+) from intracellular storage organelles in response to extracellular and intracellular stimuli. Consequently, these large, tetrameric proteins play a central role in Ca(2+) signalling and Ca(2+) homeostasis in virtually all cells. Recent data suggests that intracellular Ca(2+) release channels may also have an important pathophysiological function in certain disease states, including cardiac arrhythmias and heart failure. As a result, there has been much interest in the identification and characterization of novel, selective regulators of these channels. In this article, we review the wide array of pharmacological agents that interact directly with intracellular Ca(2+) release channels and describe the mechanisms underlying their ability to modify channel function.     

Inhibition of vasodilator neurotransmission in the sheep middle cerebral artery by VIP antiserum

1 Middle cerebral artery rings from the sheep were relaxed by vasoactive intestinal peptide (VIP) (20–200 nM ) or by stimulation of the vasodilator nerves, electrical field stimulation (EFS). 2 VIP antiserum 1:256 inhibited the relaxation produced by both exogenous VIP (from 70 ± 5 to 32 ± 9% of 5-HT-induced tone at 200 nM VIP) and by EFS (from 43 ± 5 to 26 ± 6% of 5-HT-induced tone after 20 min), while pre-immune serum was inactive. 3 Capsaicin (1 μm ) produced a transient relaxation but did not alter the response to EFS which was subsequently inhibited by addition of L -NOArg (100 μM ). VIP-induced relaxation was antagonized by L -NOArg (50 μM ) (from 68 ± 5 to 46 ± 2% relaxation of 5-HT-induced tone) but not by D -NOArg. 4 Exogenous VIP produced an approximately 2.4-fold increase in cyclic GMP content which was prevented by preincubation with L -NOArg (100 μM ) but not D -NOArg. 5 VIP and neuronal NOS immunoreactivity was co-localized to the same adventitial nerve fibres in the sheep middle cerebral artery. 6 These results provide evidence that neurogenic relaxation in the sheep middle cerebral artery is, at least in part, mediated by VIP, involving activation of NO synthase.     

Cannabinoids produce neuroprotection by reducing intracellular calcium release from ryanodine-sensitive stores

Exogenously administered cannabinoids are neuroprotective in several different cellular and animal models. In the current study, two cannabinoid CB1 receptor ligands (WIN 55,212-2, CP 55,940) markedly reduced hippocampal cell death, in a time-dependent manner, in cultured neurons subjected to high levels of NMDA (15 μM). WIN 55,212-2 was also shown to inhibit the NMDA-induced increase in intracellular calcium concentration ([Ca²⁺]_i) indicated by FURA-2 fluorescence imaging in the same cultured neurons. Changes in [Ca²⁺]_i occurred with similar concentrations (25–100 nM) and in the same time-dependent manner (pre-exposure 1–15 min) as CB1 receptor mediated neuroprotective actions. Both effects were blocked by the CB1 receptor antagonist SR141716A. An underlying mechanism was indicated by the fact that (1) the NMDA-induced increase in [Ca²⁺]_i was inhibited by ryanodine, implicating a ryanodine receptor (RyR) coupled intracellular calcium channel, and (2) the cannabinoid influence involved a reduction in cAMP cAMP-dependent protein kinase (PKA) dependent phosphorylation of the same RyR levels that regulate channel. Moreover the time course of CB1 receptor mediated inhibition of PKA phosphorylation was directly related to effective pre-exposure intervals for cannabinoid neuroprotection. Control studies ruled out the involvement of inositol-trisphosphate (IP3) pathways, enhanced calcium reuptake and voltage sensitive calcium channels in the neuroprotective process. The results suggest that cannabinoids prevent cell death by initiating a time and dose dependent inhibition of adenylyl cyclase, that outlasts direct action at the CB1 receptor and is capable of reducing [Ca²⁺]_i via a cAMP/PKA-dependent process during the neurotoxic event.     

Noradrenaline release induced by ouabain and vanadate in cat cerebral and peripheral arteries

The effects of 10(-4) M ouabain and 10(-3) M vanadate (Na3VO4) on [3H]noradrenaline release from cat cerebral and femoral arteries was studied. Ouabain induced tritium secretion in cerebral arteries, but not in femoral ones, which was reduced by Ca suppression and potentiated by extracellular Na reduction to 11.9 mM. However, vanadate evoked tritium release from both kinds of vessels was unaffected under these experimental conditions. These data suggest: ouabain elicited secretion from adrenergic nerve endings is likely due to inhibition of the Na, K-ATPase and subsequent Ca influx through Na-Ca exchange, and vanadate action is mediated by another mechanism different to the Na pump blockade.     

Effects of dobutamine on isolated canine cerebral, coronary, mesenteric, and renal arteries

The effects of dobutamine on helical strips of isolated canine cerebral, coronary, mesenteric, and renal arteries was investigated. Dobutamine contracted only renal arterial strips under resting condition. When renal and mesenteric arterial strips were partially contracted with prostaglandin F2 alpha (PGF2 alpha), dobutamine caused further concentration-related contraction, while coronary arterial strips were relaxed. Cerebral arterial strips, on the other hand, did not significantly respond to dobutamine. After treatment with 10(-5) M dl-phenoxybenzamine hydrochloride (POB) for 1 h, dobutamine-induced contractions of partially precontracted mesenteric and renal arterial strips were converted to relaxations. Relaxations of coronary arteries were not potentiated by the alpha-antagonist, but were attenuated by treatment with 10(-6) M propranolol and 10(-6) M metoprolol to a similar extent. On the other hand, relaxations of mesenteric and renal arterial strips were not inhibited by metoprolol but by propranolol. Droperidol (3 X 10(-5) M) failed to significantly alter the concentration-response curve for dobutamine. These results suggest that dobutamine causes vasoconstriction mediated by apha-adrenergic receptor and vasodilatation mediated by beta 1- and beta 2-adrenoceptors. Dobutamine does not appear to act on dopamine receptors.     

Cellular calcium and the contraction induced by prostaglandin F2 alpha in feline cerebral arteries

The influences of different calcium-entry blockers, sialidase and caffeine on the biphasic contraction induced by prostaglandin (PG) F2 alpha in the feline basilar artery (BA) were studied in calcium-free medium. After incubation in calcium-free solution, PGF2 alpha induced a contraction of the BA amounting to 87% of the contraction in calcium-containing solution. The response was biphasic in 41 out of 42 vessel segments. PGF2 alpha-induced contractions were markedly attenuated in TRIS-buffered solutions as compared to contractions in Krebs solution. PGF2 alpha failed to induce a biphasic contraction (8 out of 9 preparations) in calcium-free HEPES-buffered solution. Calcium entry blockade with 1 mM manganese or 10(-5) M diltiazem abolished the second and major phase of the PGF2 alpha-induced contraction in calcium-free Krebs solution. The second contraction phase was also eliminated in four out of five preparations pretreated with sialidase (1 unit/ml for 30 min.), but was unaffected by a brief exposure to 20 mM caffeine in calcium-free medium. The present findings strongly support previous suggestions that a major part of the PGF2 alpha-induced contraction in calcium-free medium is mediated via the release of calcium bound to the exterior aspect of the cell membrane.     

Pharmacological characterization of postjunctional alpha-adrenoceptors in cerebral arteries from the sheep.

1. The responsiveness to noradrenaline was characterized in cerebral arteries from the sheep, since this species was large enough to permit a comparison of arteries from different parts of the cerebral vasculature. 2. Noradrenaline caused contraction of the basilar artery, middle cerebral artery and small pial arteries by stimulation of alpha 1-adrenoceptors. 3. The maximum contraction to noradrenaline was small in the basilar artery (28% of the 5-hydroxytryptamine (5-HT) maximum) but larger in the middle cerebral artery (78% of the 5-HT maximum) and pial artery (92% of the 5-HT maximum) of the sheep. 4. Cocaine (10 microM) potentiated noradrenaline-induced contractions in the sheep middle cerebral artery but not in the sheep basilar artery. 5. The noradrenaline contraction, relative to the 5-HT contraction, was not affected by removal of the endothelium in either the sheep basilar or middle cerebral artery. 6. The results showed a variation within the sheep cerebral vasculature in the response to noradrenaline which cannot be explained by regional differences in alpha-adrenoceptor subtypes, noradrenaline uptake mechanisms or endothelial function.     

EP4 prostanoid receptor-mediated vasodilatation of human middle cerebral arteries

1. Dilatation of the cerebral vasculature is recognised to be involved in the pathophysiology of migraine. Furthermore, elevated levels of prostaglandin E(2) (PGE(2)) occur in the blood, plasma and saliva of migraineurs during an attack, suggestive of a contributory role. In the present study, we have characterised the prostanoid receptors involved in the relaxation and contraction of human middle cerebral arteries in vitro. 2. In the presence of indomethacin (3 microm) and the TP receptor antagonist GR32191 (1 microM), PGE(2) was found to relax phenylephrine precontracted cerebral arterial rings in a concentration-dependent manner (mean pEC(50) 8.0+/-0.1, n=5). 3. Establishment of a rank order of potency using the EP(4)>EP(2) agonist 11-deoxy PGE(1), and the EP(2)>EP(4) agonist PGE(1)-OH (mean pEC(50) of 7.6+/-0.1 (n=6) and 6.4+/-0.1 (n=4), respectively), suggested the presence of functional EP(4) receptors. Furthermore, the selective EP(2) receptor agonist butaprost at concentrations <1 microM failed to relax the tissues. 4. Blockade of EP(4) receptors with the EP(4) receptor antagonists AH23848 and EP(4)A caused significant rightward displacements in PGE(2) concentration-response curves, exhibiting pA(2) and pK(B) values of 5.7+/-0.1, n=3, and 8.4, n=3, respectively. 5. The IP receptor agonists iloprost and cicaprost relaxed phenylephrine precontracted cerebral arterial rings (mean pEC(50) values 8.3+/-0.1 (n=4) and 8.1+/-0.1 (n=9), respectively). In contrast, the DP and FP receptor agonists PGD(2) and PGF(2 alpha) failed to cause appreciable relaxation or contraction at concentrations of up to 30 microm. In the absence of phenylephrine contraction and GR32191, the TP receptor agonist U46619 caused concentration-dependent contraction of cerebral artery (mean pEC(50) 7.4+/-0.3, n=3). 6. These data demonstrate the presence of prostanoid EP(4) receptors mediating PGE(2) vasodilatation of human middle cerebral artery. IP receptors mediating relaxation and TP receptors mediating contraction were also functionally demonstrated.     

Influence of calcium on noradrenaline release evoked by 5-hydroxytryptamine,tyramine and potassium from goat pial arteries

The release of tritium (³ H) evoked by tyramine, potassium (K⁺) and 5-hydroxytryptamine (5-HT) from goat pial arteries preloaded with [³ H]noradrenaline (³ H-NA) was studied. In normal Krebs-bicarbonate solution (KBS) all these agents caused a transient increase in radioactivity release over the basal spontaneous outflow. The pattern of release evoked by 5-HT was similar to that induced by tyramine with a slow onset and decline, but different from that induced by K⁺ which produced a rapid peak of ³ H release followed by a quick fall. The removal of Ca²⁺ from the medium did not modify the efflux of radioactivity caused by tyramine, but the ³ H efflux produced by K⁺ was markedly reduced. Nevertheless, in this Ca²⁺ -free medium the ³ H release evoked by 5-HT was partially, but significantly, decreased. These results indicate that K⁺ evokes NA release by a Ca²⁺ -dependent process, probably of an exocytotic nature, while tyramine mediates NA release by means of a Ca²⁺ -independent mechanism. However, 5-HT possesses a Ca²⁺ -dependent and a tyramine-like component.     

Effect of MCI-176, a new calcium antagonist, on the calcium induced contraction of isolated porcine coronary arteries

Calcium antagonistic activity of MCI-176, a new calcium antagonist, was compared with those of diltiazem and nifedipine in isolated depolarized porcine coronary arteries. MCI-176, diltiazem and nifedipine competitively inhibited calcium contraction of the large coronary arteries, and their pA2 values were 7.49, 6.89 and 9.55, respectively. Similar competitive inhibition by MCI-176, diltiazem and nifedipine of calcium contraction was also observed in the small coronary arteries, and their pA2 values were 7.38, 6.83 and 9.91, respectively. Although calcium antagonistic activity of nifedipine was several hundreds times more potent than MCI-176 and diltiazem, the action of nifedipine, unlike MCI-176 and diltiazem, favored the small coronary arteries rather than the large coronary arteries.     

Endothelium-dependent relaxations in sheep pulmonary arteries and veins: resistance to block by NG-nitro-L-arginine in pulmonary hypertension.

1. The effect of the nitric oxide synthase inhibitor, NG-nitro-L-arginine (L-NOARG), on endothelium-dependent relaxation to a receptor-independent agent, ionomycin, was examined in isolated pulmonary arteries and veins from control, short-term and chronic pulmonary hypertensive sheep. All vessel segments were contracted to optimal levels of active force with endothelin-1 to record endothelium-dependent relaxation. 2. Pulmonary hypertension was induced by continuous pulmonary artery air embolization for 1 day (short-term) and 14 days (chronic) and was associated with a 2 and 3 fold increase in pulmonary vascular resistance respectively. 3. L-NOARG (0.1 mM) reduced the maximum relaxation (Rmax) to ionomycin in large and medium-sized pulmonary arteries from control sheep by approximately 70%. By contrast, L-NOARG (0.1 mM) did not inhibit the Rmax to ionomycin in matched vessels from short-term and chronic pulmonary hypertensive sheep. 4. Resistance of ionomycin-induced relaxations to inhibition by L-NOARG, was confined to the arterial vasculature in chronic pulmonary hypertensive animals, as relaxations to ionomycin in large and medium-sized chronic pulmonary hypertensive veins were, like those in control veins, abolished by L-NOARG. Both large and medium-sized pulmonary veins from short-term pulmonary hypertensive sheep, however, were resistant to block by L-NOARG. 5. Neither sensitivity (pEC50) nor Rmax to ionomycin in large, short-term pulmonary hypertensive arteries was affected when the extracellular concentration of K+ was increased isotonically to 30 mM. Nifedipine (0.3 microM) was present throughout to prevent high K(+)-induced smooth muscle contraction. In the presence of this high extracellular K+, however, L-NOARG (0.1 mM) caused complete inhibition of the relaxation to ionomycin, whereas in normal extracellular K+ (4.7 mM), L-NOARG only weakly inhibited ionomycin relaxations. 6. In conclusion, the onset of pulmonary hypertension in sheep following air embolization, is associated with the development of resistance of endothelium-dependent relaxations to block by L-NOARG. The mechanism of L-NOARG resistance appears to be due to the up-regulation of a K+ channel-mediated backup vasodilator mechanism which can compensate for the loss of nitric oxide (NO)-mediated relaxation. Although this mechanism remains functionally 'silent' in the presence of NO it is able to maintain adequate endothelium-dependent vasodilatation during pulmonary hypertension if NO synthesis is compromised.     

Heterogeneous responses to vasodilators of dog proximal and distal middle cerebral arteries

Responses to various vasodilators were compared in helical strips of proximal and distal middle cerebral arteries isolated from the same dogs, which were partially precontracted with prostaglandin (PG) F2 alpha or K+. Nicotine-induced relaxations, selectively blocked by hexamethonium, were greater in the proximal than in the distal portion, whereas K+ (5 mM)-induced relaxations, reversed to contractions by ouabain, were greater in the distal portion. Angiotensin II and PGI2 relaxed distal middle cerebral arteries to a greater extent; the difference in relaxations induced by the octapeptide in large and small arteries was more evident. Relaxations induced by isoproterenol and adenosine were greater in distal arteries; in contrast, those induced by nitroglycerin were greater in proximal arteries. Acetylcholine and verapamil relaxed the arteries of both portions to a similar extent. It may be concluded that proximal middle cerebral artery functions are regulated by dilator nerves more intensely than the distal artery functions, and the electrogenic Na+ pump in cell membrane of distal middle cerebral arteries leaves more room for being activated under the experimental conditions used. Angiotensin II appears to liberate more PGI2 from the distal cerebral arteries.     

Responses of sheep and kitten isolated coronary arteries to some vasoactive agents

In the present study, the effects of a range of vasoactive agents were assessed in sheep isolated coronary artery strips and in kitten isolated perfused coronary arteries. Both preparations contained muscarinic cholinoceptors. However, these receptors mediated contraction of sheep coronaries while dilatation was seen in the kitten. In sheep arteries, 5-hydroxytryptamine caused increases in resting tone and relaxation of acetylcholine-induced tone, while vasoconstriction was the predominant response in the kitten. Angiotensin increased tone in both preparations, although tachyphylaxis was evident. Conversely, adenosine and ATP caused reductions in tone in both preparations. Alpha-adrenoceptor agonists caused vasodilatation in kitten coronaries and decreased acetylcholine-induced tone in sheep arteries. However, weak contractions were observed in sheep coronaries in the absence of induced tone. Results also indicate that sympatomimetic amines mediate relaxation of sheep coronary arteries via beta 1-adrenoceptors. Sheep coronaries are readily and inexpensively obtained and provide a useful tool for the further study of coronary vascular smooth muscle.     

Phorbol 12,13-dibutyrate increases vascular tone but has a dual action on intracellular calcium levels in porcine coronary arteries

Summary The effects of phorbol 12,13-dibutyrate (PDBu), a protein kinase C activator, on the tone and intracellular calcium level (Ca _infi ^sup2+ ) of vascular smooth muscles were simultaneously measured by use of a force-displacement transducer and the fura-2 microscopic fluorometric technique in porcine coronary arteries. Cumulatively applied PDBu produced a slowly developing and concentration-dependent contraction in the concentration range of 10^-8–10^-6 mol/l. Contractions induced by PDBu were sustained after removal of PDBu. Changes in Ca _infi ^sup2+ produced by PDBu were very slight, although Ca _infi ^sup2+ was increased at lower concentrations (3 × 10^–7 and 10^–7 mol/l) and decreased at higher concentrations (3 × 10^–7 and 10^–7 mol/l). Verapamil 10^–7 mol/l, partially inhibited the contractions throughout the concentration range of PDBu and the increase in Ca _infi ^sup2+ induced by lower concentrations of PDBu. When the effects of PDBu were compared with those of the 90 mmol/l KCl medium, the force of contraction induced by a single concentration of 10^–7 mol/l, PDBu was about 50% and the increase in Ca _infi ^sup2+ was about 10%. Removal of extracellular calcium by 1 mmol/l EGTA decreased Ca _infi ^sup2+ by about 20% but vascular tone did not change. PDBu (10^–7 mol/l) produced a small contraction without an increase in Ca _infi ^sup2+ in the Ca²⁺-free medium. Depolarization by the 20 mmol/l KCl medium increased Ca _infi ^sup2+ by about 20%, whereas vascular tone did not change. The contraction and increase in Ca _infi ^sup2+ induced by 10^–7 mol/l, PDBu were both enhanced in the 20 mmol/l KCl medium. The contraction induced by 10^–7 mol/l, PDBu was more than 100%, whereas Ca _infi ^sup2+ decreased by 10–20%. Contractions induced by 10^–7 mol/I PDBu were partially inhibited in the Ca _infi ^sup2+ -free medium and were not potentiated in the 20 mmol/l KCl medium. These results suggest that activation of protein kinase C by phorbol ester has a dual action on Ca _infi ^sup2+ : a slight increase and a slight decrease. The contractions induced by phorbol ester not only depend on an increase in Ca _infi ^sup2+ but involve the sensitization of contractile system to calcium or the generation of force via a Ca²⁺-independent process. Send offprint requests to N. Taira at the above address     

超低分子量肝素抑制神经细胞内钙释放保护谷氨酸损伤原代培养大鼠神经元

目的探讨超低分子量肝素(ultra low molecular weight heparin,ULMWH)对谷氨酸(Glu)诱导原代培养大鼠大脑皮层神经细胞损伤的保护作用及其作用机制。方法采用体外培养大鼠大脑皮层神经细胞,建立谷氨酸(Glu)诱导损伤模型,采用MTT法检测细胞活力、Hoechest33258染色法观察凋亡细胞形态改变和检测凋亡细胞数。同时,采用Fura-2/AM双波长荧光分光光度法测定神经细胞内钙离子浓度([Ca2+]i)。结果提前应用ULMWH可提高Glu损伤神经细胞的生存能力,降低Glu诱导的凋亡细胞数。同时,ULM-WH不论是在含Ca2+测量介质还是在无Ca2+测量介质中均可降低神经细胞[Ca2+]i。结论ULMWH对Glu损伤神经细胞具有保护作用,该作用可能与其抑制神经细胞内Ca2+释放而降低胞内[Ca2+]i有关。     

Heparin-insensitive calcium release from intracellular stores triggered by the recombinant human parathyroid hormone receptor.

1. In the present study we have characterized the parathyroid hormone (PTH)-induced calcium signalling in 293 cells stably transfected with the human PTH receptor cDNA. In these cells, human PTH-1(1-38) strongly stimulates adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation (EC50 = 0.39 nM) but fails to activate phosphoinositide (PI) turnover. The latter pathway is strongly activated, however, by carbachol (CCh) acting through endogenous M3-muscarinic receptors. 2. Despite the lack of detectable inositol phosphate (IP) formation, hPTH-(1-38) elicited calcium transients (EC50 = 11.2 nM) which were comparable to the signals evoked by CCh. These signals are independent of cyclic AMP generation as cyclic AMP elevating agents did not mimic or modify the PTH response. 3. The PTH-stimulated calcium signal still occurred in calcium-free medium but was absent in cells pretreated with thapsigargin, an inhibitor of the calcium pump of the endoplasmic reticulum (ER). hPTH-(1-38) did not accelerate Mn(2+)-influx through the plasma membrane. These data indicate that PTH releases calcium from intracellular stores. 4. Using heparin, an inhibitor of the IP3-activated calcium release channel of the ER, we tested whether the formation of a low amount of IP3, escaping detection by our biochemical assay, might be the origin of the PTH-induced calcium response. However, intracellular infusion of heparin through patch pipettes in voltage clamp experiments failed to block hPTH-(1-38)-induced calcium signals, whereas it abolished the CCh response.(ABSTRACT TRUNCATED AT 250 WORDS)