Mechanism of CGRP-induced relaxation in rat intramural coronary arteries.

1. This study investigates the mechanism of CGRP-induced relaxation in intramural coronary arteries by determining the effect of CGRP on cytosolic Ca(2+) concentration ([Ca(2+)](i)) using FURA-2 technique. 2. CGRP concentration-dependently (10 pM - 100 nM) decreased the [Ca(2+)](i) and tension of coronary arteries precontracted with either U46619 or BAY K 8644, and also of resting coronary arteries in PSS. In 36 mM K(+)-depolarized arteries, CGRP reduced only the tension without affecting the [Ca(2+)](i). 3. In 300 nM U46619- precontracted arteries, pretreatment with 10 microM thapsigargin significantly (P<0.05) attenuated the CGRP-induced reduction in the tension (but not [Ca(2+)](i)). 4. In 300 nM U46619-precontracted arteries, pretreatment with either 100 nM charybdotoxin or 100 nM iberiotoxin or 10 nM felodipine significantly (P<0.05) attenuated the CGRP-induced reduction in both [Ca(2+)](i) and tension. In contrast, 1 microM glibenclamide did not affect the CGRP-induced responses in these coronary arteries. 5. In resting coronary arteries, only pretreatment with the combination of 1 microM glibenclamide and 100 nM charybdotoxin attenuated the CGRP-induced decrease in the [Ca(2+)](i) and tension, suggesting a different mechanism of action for CGRP in resting coronary arteries. 6. We conclude that CGRP relaxes precontracted rat coronary arteries via three mechanisms: (1) a decrease in [Ca(2+)](i) by inhibiting the Ca(2+) influx through membrane hyperpolarization mediated partly by activation of the large conductance Ca(2+)-activated potassium channels, (2) a decrease in [Ca(2+)](i) presumably by sequestrating cytosolic Ca(2+) into thapsigargin-sensitive Ca(2+) storage sites and (3) a decrease in the Ca(2+)-sensitivity of the contractile apparatus. In resting coronary arteries, however, there seems to be an interplay between different types of K(+) channels.

Various nitric oxide donors protect chick embryonic neurons from cyanide-induced apoptosis.

The discovery of numerous biochemical effects of cyanide not directly related to the inhibition of the respiratory chain, including the involvement of apoptosis, has challenged the basis of traditional antidote treatment, which primarily depends on nitrite-induced conversion of hemoglobin into methemoglobin, releasing the blockade of cytochrome c oxidase by high-affinity binding of cyanide as cyanmethemoglobin. The fact that amyl nitrite has antidotal effects not related to methemoglobin formation has unfolded new mechanism of actions of nitrites including release of nitric oxide (NO). In this study, we characterized the effect of various NO donor compounds on cyanide-induced cell death in cultured chick embryonic neurons. Apoptosis was induced by treating the neuronal cultures with 1 mM NaCN for 1 h, followed by a cyanide-free incubation period of 23 h. Using this treatment protocol, we showed that cyanide-induced apoptosis was blocked in the presence of the different NO donors sodium nitroprusside, S-nitrosoglutathione, S-nitroso-N-acetylpenicillamin, nitroglycerin, 3-morpholinosydnonimine, and diethylamine nitric oxide, indicating independence of the redox-related species of NO released. The effect was confirmed to be mediated by NO, since exhausted NO donors did not afford protection, and the mechanism likely involved chemical modification of thiol groups, since the effect was completely reversed by dithiothreitol. Furthermore, NMDA antagonists protected against cyanide-induced cell death, whereas inhibitors of nitric oxide synthase increased cyanide-induced apoptotic damage, indicating a protective effect of endogenously generated NO, at least in cell cultures.

Non-competitive antagonism of amylin on CGRP(1)-receptors in rat coronary small arteries.

We examined the interaction between rat-amylin and relaxations induced by rat-alphaCGRP and isoprenaline in rat isolated coronary small arteries. Amylin, 0.1 - 100 nM, had a concentration dependent non-competitive antagonistic effect on rat-alphaCGRP-induced responses with an EC(50) of approximately 1 nM. Amylin did not affect the relaxations induced by isoprenaline at a concentration of 10 nM. The apparent equilibrium dissociation constant, K(A), for CGRP(1)-receptors in the rat coronary small arteries was approximately 2 nM. Analysis of the relationship between receptor occupancy and response to rat-alphaCGRP indicates that the receptor reserve is small. Our results show that amylin in low concentrations acts as a selective non-competitive inhibitor at CGRP(1)-receptors in rat isolated coronary small arteries.

The effect of long-term streptozotocin-induced diabetes on contractile and relaxation responses of coronary arteries: selective attenuation of CGRP-induced relaxations.

1. This study investigates the effect of partially metabolic controlled long-term (34 weeks) streptozotocin (STZ)-induced diabetes on relaxation and contractile responses of isolated coronary arteries to seven different vasoactive agents. 2. The average fasting and non-fasting blood glucose concentrations (mM) were significantly elevated in STZ-induced diabetic rats (P<0.0001; 10.4+/-0.4 and 16. 6+/-1.1, n=15) compared to those (4.3+/-0.03 and 4.7+/-0.18, n=11) in age-matched controls. The level of glycated haemoglobin (HbA(1)) was also significantly (P<0.0001) increased in STZ-induced diabetic rats. In STZ-induced diabetic rats, the HbA(1) levels were significantly correlated with the non-fasting blood glucose concentrations (r=0.76; P=0.003; n=13). In both groups, there was no significant correlation between the HbA(1) levels and maximal responses or sensitivities to the vasoactive agents. 3. The maximal relaxation induced by rat-alphacalcitonin gene-related peptide (rat-alphaCGRP) was significantly attenuated in the coronary arteries of STZ-induced diabetic rats (P<0.05; 40+/-7%, n=15) compared to that in age-matched controls (63+/-3%, n=11). However, there was no significant difference in the sensitivity to rat-alphaCGRP between the two groups. 4. There was no significant difference in either maximal response or sensitivity to any of the six other vasoactive agents between STZ- induced diabetic rats (n=15) and age-matched controls (n=11). 5. Our results show that partially metabolic controlled long-term (34 weeks) STZ-induced diabetes causes a selective depression of rat-alphaCGRP-induced relaxation in the intramural coronary arteries of Wistar rats.

Coronary endothelial dysfunction of isolated hearts subjected to prolonged cold storage: patterns and contributing factors.

BACKGROUND: A key role of endothelial dysfunction in the pathogenesis of early low coronary flow of heart transplants and late cardiac allograft vasculopathy indicates the importance of optimal coronary endothelial preservation during cold heart storage. We designed this study to investigate the effect of prolonged cold storage on endothelial and smooth muscle function of proximal (epicardial) and distal (intramyocardial) coronary arteries. METHODS: Four groups of isolated rat hearts were subjected to cold cardioplegic perfusion and immersed in storage medium at 4 degrees C. In groups 1, 2, and 3, hearts were perfused with and stored in Celsior solution for 10, 15, and 30 hours, respectively. In group 4, hearts were perfused with Plegisol and stored in saline for 15 hours. At the end of cold heart storage, arterial segments were taken from the proximal and distal parts of the left coronary artery and mounted on an isometric wire myograph for functional studies. In fifth group, proximal and distal segments of coronary artery isolated from fresh hearts were used as controls. At the end of control measurements, these vessels were used for storage in vitro at 4 degrees C for 15 hours in saline (group 5A) or Celsior (group 5B). RESULTS: The endothelium-dependent relaxation to acetylcholine was reduced in distal coronary arteries in group 1, and in both proximal and distal coronary artery segments in groups 2, 3, 4, and 5A. Endothelial function was significantly more impaired in both proximal and distal coronary arteries in group 4, as compared with group 2. The impairment of relaxation to acetylcholine was more pronounced following cold storage of the heart than after a similarly long storage of the isolated vessels. The endothelium-independent relaxations to isoprenaline did not differ among all groups. The basal myogenic tone was increased in distal coronary arteries in group 1, and in both proximal and distal coronary arteries in groups 2, 3, 4, and 5A. The sensitivity to the vasoconstricting action of 5-hydroxytryptamine was increased in distal coronary arteries in group 2, and in both proximal and distal coronary arteries in groups 3, 4, and 5A. CONCLUSIONS: Prolonged ischemic cold heart storage induces coronary endothelial dysfunction that is more pronounced in distal than in proximal arteries and is related to the duration of heart storage and the composition of storage medium.

Caliber dependent calcitonin gene-related peptide-induced relaxation in rat coronary arteries: effect of K+ on the tachyphylaxis.

The influence of vessel caliber on rat calcitonin gene-related peptide (rat-alphaCGRP)-induced responses and the reproducibility of rat-alphaCGRP concentration-response curves were investigated in the left intramural coronary artery of Sprague-Dawley rats. Rat-alphaCGRP (10(-11)-10(-7) M) induced concentration-dependent relaxations with a pD2-value equal to 8.43 +/- 0.05 (n = 44) and maximal relaxation equal to 52 +/- 3% (n = 44). Both the maximal relaxation and the sensitivity to rat-alphaCGRP were significantly and inversely correlated with vessel lumen diameter. The coronary arteries developed tachyphylaxis in response to rat-alphaCGRP, which was concentration dependently decreased by activating the vessels twice with a buffer containing 36 or 125 mM K+. The rat-alphaCGRP-curve became fully reproducible after activation of the arteries twice with 125 mM K+. These results indicate a caliber-related dependency of both the effect of and sensitivity to rat-alphaCGRP in intramural rat coronary arteries because the arteries become more sensitive and reactive to rat-alphaCGRP with decreasing caliber. Tachyphylaxis can be avoided by repeated activation with 125 mM K+.

Characterization of calcitonin gene-related peptide (CGRP) receptors in intramural coronary arteries from male and female Sprague Dawley rats.

1. In this study we characterized the CGRP-receptor subtype by Schild-plot analysis using the C-terminal fragment, human-alphaCGRP(8-37), a putative competitive CGRP1-receptor selective antagonist. In addition, the effect of rat-alphaCGRP was compared with that of homologous peptides rat-betaCGRP, rat-amylin, rat-adrenomedullin and [Cys(Acm)2,7]-human-alphaCGRP, a putative selective CGRP2-receptor agonist, in the left coronary arteries of 3 months old male and female Sprague Dawley rats. 2. Isolated rings from the distal, intramural part of the left anterior descending (LAD) coronary artery in both groups of rats were mounted on a double wire-myograph. The arteries were then stretched to their optimal lumen diameter for active tension development and precontracted with 10(-5) M prostaglandin F2alpha (PGF2alpha), after which agonists were added to the organ bath in a cumulative manner. 3. Rat-alphaCGRP induced endothelium-independent relaxations in male and female Sprague-Dawley rats. Rat-betaCGRP concentration-response relations (10[-11]-10[-7] M) were similar to those of rat-alphaCGRP in either sex. The maximal relaxations induced by rat-amylin and rat-adrenomedullin, both at 10(-6) M, were significantly (P<0.05) lower than those induced by rat-alpha- and rat-betaCGRP. In contrast, the selective CGRP2-receptor agonist [Cys(Acm)2,7]-human-alphaCGRP failed to induce significant relaxations at the highest concentration used (10[-7] M) in the coronary arteries of male and female rats. 4. The C-terminal fragment, human-alphaCGRP(8-37) blocked concentration-dependently (10[-7]-10[-6] M) the rat-alphaCGRP-induced relaxation in 10(-5) M PGF2alpha-precontracted coronary arteries. The slopes of the regression lines of the Schild-plots for both male and female rats were not significantly (P>0.05) different from unity and the pA2 values for human-alphaCGRP(8-37) were 6.93 and 6.98 in arteries from male and female rats, respectively. There was no significant (P>0.05) difference in estimated pKB values for human-alphaCGRP(8-37) between male (6.99+/-0.10, n=13) and female (6.95+/-0.08, n=13) rats. 5. The concentration-response relationships for rat-alpha- and rat-betaCGRP were similar in male and female Sprague Dawley rats. The predominant CGRP receptor subtype in small intramural coronary arteries appeared to belong to the CGRP1-receptor subtype in both sexes.

Pharmacological characterization of coronary small arteries from pigs with chronic ischaemic myocardial remodelling.

1. The effect of chronic ischaemic myocardial remodelling on small coronary artery reactivity in vitro was studied in a newly developed pig model. 2. Pigs were subjected to selective intracoronary embolizations with microspheres in the left anterior descending artery and circumflex artery causing scattered myocardial fibrosis. After an observation period of 130 days, heart dimensions and ejection fraction were determined with magnetic resonance imaging. Small arteries were isolated from the left ventricle and mounted as ring preparations in a myograph. Control arteries were taken from matched non-embolized pigs. 3. Compared with control pigs, end-systolic and diastolic volumes increased and left ventricular mass nearly doubled in embolized pigs. This indicates substantial myocardial hypertrophy, as the fraction area of fibrosis was only 12%. 4. Coronary small arteries preconstricted with 30 mmol/l KCI showed a normal contractile response to acetylcholine and 5-hydroxytryptamine. Sensitivity of the relaxation to bradykinin was nearly 3-fold increased and also slightly enhanced to isoprenaline in arteries from embolized pigs compared with controls, whereas relaxation to 5-hydroxytryptamine in the presence of ketanserin was similar. After inhibition of nitric oxide synthase with NG-nitro-L-arginine the sensitivity to acetylcholine increased to a similar extent in arteries from embolized pigs and controls. NG-Nitro-L-arginine abolished the relaxing effects of bradykinin and of 5-hydroxytryptamine in the presence of ketanserin. 5. We conclude that both the contractile function of the smooth muscle cells and the endothelial production or action of nitric oxide is preserved or slightly enhanced in coronary small arteries from pigs with chronic myocardial remodelling.

Protective effect of reduced glutathione on endothelial function of coronary arteries subjected to prolonged cold storage.

BACKGROUND: Endothelial dysfunction can play a key role in early no reflow and late accelerated cardiac graft arteriosclerosis. We studied the direct effect of reduced glutathione (GSH) on the preservation of endothelial function of coronary arteries subjected to prolonged cold storage. METHODS: Ring preparations were dissected from rat left coronary arteries and mounted on an isometric wire myograph. After control measurements, artery segments were exposed to 15 hr of hypothermic (+4 degrees C) incubation in either normal saline (group 1), GSH-free Celsior solution (a new heart preservation solution) (group 2), or Celsior solution with 3 mmol/L of GSH (group 3). RESULTS: After storage, the basal tone was increased in groups 1 and 2, but it did not change in group 3. The endothelium-dependent relaxation to acetylcholine was reduced in groups 1 and 2, but it was not affected in group 3. The sensitivity to acetylcholine was decreased in group 1, and there were no changes in groups 2 and 3. CONCLUSIONS: Our data suggest that fresh GSH in Celsior solution improves preservation of coronary endothelial function.

Porcine coronary artery pharmacodynamics in vitro evaluated by a new intravascular technique: relation to axial stretch.

A new intravascular balloon catheter-based technique, impedance planimetry and the wire-mounted isometric tension technique commonly employed to study vessel pharmacodynamics in vitro, were compared. Porcine left anterior descendent coronary artery reactivity to nifedipine was assessed and the influence of 20% axial stretch was investigated. There were no histological differences between segments where the impedance planimetry balloon had been inflated and untouched segments. EC50 values differed significantly between the three procedures applied: The isometric method (n = 7): 2.54 +/- 0.44.10(-9) M; nonstretched arteries by the impedance planimetric method (n = 7): 1.99 +/- 0.40.10(-8) M; arteries 20% axially stretched (n = 7): 2.00 +/- 1.36.10(-7)M (isometric and nonstretched: p < 0.05; isometric and stretched: p < 0.001; nonstretched and stretched: p < 0.05). Maximal relaxant responses to nifedipine were 91.8 +/- 2.1% (isometric method), 105.1 +/- 2.3% (nonstretched), and 104.9 +/- 7.7% (stretched) (ANOVA, p = 0.11). In stretched arteries, the initial 12-min response to an increased dose of nifedipine was more rapid than the response of nonstretched arteries at a concentration of 1.10(-7) M (p = 0.038) and had a nonsignificant tendency toward a more rapid response at other concentrations. Resting tone could not be demonstrated and time control experiments showed no change in the maximal vessel response to potassium with any of the three methods. A new method in the evaluation of artery pharmacodynamics in vitro was presented. The study demonstrated that axial stretching of an artery has impact on the pharmacodynamic reactivity to nifedipine in porcine coronary arteries. Further studies are needed to evaluate the impact of the method on endothelial function.

Effects of doxazosin on functional alterations of isolated coronary arteries from cholesterol-fed rabbits.

Anti-hypertensive treatment is much less successful at reducing coronary artery disease than at reducing mortality from stroke and congestive heart failure. The effects of the alpha-adrenergic antagonist doxazosin on progression of atheromatous lesions and functional responses of isolated coronary arteries from cholesterol-fed rabbits have been investigated. Normotensive rabbits were fed either a standard chow (control, n = 8) or a 1% cholesterol-rich diet (n = 16) for 20 weeks. After 3 weeks the cholesterol-fed animals were assigned randomly to two groups either given placebo capsules (n = 8) or treated with doxazosin (5 mg kg-1 day-1; n = 8). Doxazosin reduced the mean arterial blood pressure by 10% that of the control and placebo-treated cholesterol-fed rabbits, but did not affect the plasma cholesterol, triacylglycerol and phospholipid levels, which were, after 20 weeks, severalfold increased in the cholesterol-fed rabbits compared with controls. Histological examination showed atheromatous lesions in proximal (but not distal) coronary arteries from both groups of cholesterol-fed rabbits. Doxazosin either had no effect on reduced contractions to 125 mmol L-1 potassium saline solution or increased contractions to 5-hydroxytryptamine in proximal isolated coronary arteries from the cholesterol-fed rabbits. It did, however, abolish the hyper-responsiveness of the large atheromatous coronary arteries to noradrenaline. In both vehicle-and doxazosin-treated cholesterol-fed rabbits the maximum relaxation and sensitivity to acetylcholine were significantly reduced in proximal segments compared with the control group, whereas responses to acetylcholine in distal coronary segments were not significantly different. The relaxation to sodium nitroprusside, adenosine diphosphate and isoprenaline in proximal and distal coronary arteries were similar in the three experimental groups. These results indicate that treatment of normotensive cholesterol-fed rabbits with doxazosin prevents the hyper-responsiveness to noradrenaline of proximal coronary arteries, although it does not prevent the progression of other functional alterations observed in the coronary circulation.

Regional involvement of an endothelium-derived contractile factor in the vasoactive actions of neuropeptide Y in bovine isolated retinal arteries.

1. In vitro experiments in a microvascular myograph were designed in order to investigate the effects of human neuropeptide Y (NPY), its receptor subtype and the mechanisms underlying NPY actions in bovine isolated retinal proximal (PRA) and distal (DRA) arteries. 2. A single concentration of NPY (10 nM) induced a prompt and reproducible contraction which reached a plateau within 1-4 min, after which the response returned to baseline over the next 2-10 min. Cumulative addition of NPY induced concentration-dependent contractions of bovine retinal arteries, with an EC50[M] of 1.7 nM and a maximal response equal to 54 +/- 8% of Emax (absolute maximal contractile levels of vessels) and not different from that obtained by a single addition of the peptide. There were no significant differences in either sensitivity or maximal response to NPY between PRA and DRA. 3. Porcine NPY and the selective Y1-receptor agonist, [Pro34]NPY, also induced concentration-dependent contractions of the retinal arteries with a potency and maximal response not significantly different from those of human NPY; in contrast, the selective Y2-receptor agonist, NPY(13-36), caused only a 5% contraction at the highest concentration used. 4. Removal of extracellular Ca2+ or pretreatment with the 1,4-dihydropyridine Ca(2+)-channel blocker, nifedipine (1 microM), reduced the contractile response of 10 nM NPY to 18.4 +/- 3.3% (n = 6) and 18.6 +/- 3.9% (n = 6); respectively, of the controls. 5. Mechanical removal of the endothelium depressed the maximal contraction elicited by NPY in PRA but did not affect either sensitivity or maximal response to the peptide in DRA. In endothelium-intact arteries, blockade of the cyclo-oxygenase pathway with 3 microM indomethacin increased resting tension in both PRA and DRA and significantly inhibited sensitivity and maximal contraction to NPY of PRA and DRA, respectively. The thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor antagonist, SQ30741, reduced both sensitivity and maximal contraction to NPY in PRA but not in DRA. 6. In endothelium-denuded PRA, indomethacin but not SQ30741 significantly reduced NPY maximal response and induced a marked increase in resting tension suggesting a basal release of a vasodilator prostanoid from smooth muscle cells. 7. Superoxide dismutase (SOD) (150 u ml-1) reduced the maximal contraction to NPY in PRA. Inhibition of the nitric oxide (NO) synthase with NG-nitro-L-arginine (L-NOARG) (30 microM), enhanced sensitivity and maximal contraction to NPY in both PRA and DRA. In the presence of L-NOARG, SOD did not further inhibit NPY responses in PRA. 8. NPY (10 nM) induced a 2.9 fold leftwards shift of the noradrenaline concentration-response curves in PRA and increased maximal response by 50 +/- 16%. Neither 1 nor 10 nM NPY affected noradrenaline responses in DRA. [Pro34]NPY (10 nM), but not NPY(13-36), mimicked the potentiating effect of NPY on noradrenaline responses in PRA. 9. TXA2 analogue, U46619, at 10 nM elicited 3.6 fold leftwards shift of the noradrenaline concentration-responses curves in PRA and increased the maximal contraction by 32 +/- 3%, whereas in the presence of 1 microM SQ30741, 10 nM NPY did not potentiate noradrenaline responses. 10. The present results indicate that NPY may play a role in the regulation of retinal blood flow through both a direct contractile action, independent of the vessel size and a potentiation of the responses induced by noradrenaline in the proximal part of the retinal circulation, both effects being mediated by Y1 receptors. NPY promotes Ca2+ influx through voltage-dependent Ca2+ channels and stimulates the synthesis of contractile prostanoids in PRA and DRA, although only in PRA does the peptide trigger the release of an endothelium-derived contractile factor which facilitates the contraction and also seems to account for the potentiating effect of NPY.

Beta-adrenergic receptors regulating vascular smooth muscle tone are only localized to the intraocular segment of the long posterior ciliary artery in bovine eye.

Beta-adrenergic drugs are important drugs in glaucoma treatment. Their exact mechanism of action is not yet fully understood but a decreased perfusion pressure in the ciliary body due to blockade of vasodilatory beta-adrenoceptors is thought to participate in the reduction of intraocular pressure. This study investigates the vasodilator action of beta adrenergic-agents in intra- and extraocular arteries from bovine and human eyes. Ring segments of retinal, choroidal, intraocular segment of long posterior ciliary artery, and segments of extraocular posterior ciliary arteries from bovine eyes and short posterior ciliary arteries from freshly enucleated human eyes were mounted on an isometric myograph and their reactivity to beta-adrenergic drugs were studied. Of all vessel types examined only the intraocular segment of long posterior ciliary artery precontracted with prostaglandin F2 alpha responded to 1-isoprenaline (1 nM-1 microM) with a graded concentration-dependent relaxation (32 +/- 7%, n = 10) and a pD2 of 7.5 +/- 0.2 concentrations of 1-isoprenaline greater than 1 microM induced contractions which were antagonized by a combined blockade of alpha1- and alpha2-adrenoceptors. Salbutamol (selective beta2-agonist) induced relaxations of similar magnitude as that of 1-isoprenaline but the sensitivity of the intraocular segment of long posterior ciliary arteries to salbutamol was 312 times less than that to 1-isoprenaline, pD2 7.2 +/- 0.2 and 4.4 +/- 0.1 (n = 6), respectively, whereas dobutamine (selective beta 1-agonist) had no effect between 1 nM and 10 microM. Betaxolol (selective beta 1-antagonist) and 1CI 115811 (selective beta 2-antagonist) competitively antagonized isoprenaline-induced relaxations giving pKB-values of 5.9 +/- 0.1 (n = 11) and 8.5 +/- 0.1 (n = 6), respectively. The slope of the Schild-plots were equal to unity. Human posterior ciliary arteries with spontaneous tone or tone induced by 10 microM PGF2 alpha did not react to isoprenaline (1 nM-10 microM). The bovine intraocular segment of long posterior ciliary arteries contracted to 1-noradrenaline without alpha-adrenoceptor blockade and addition of propranolol or cocaine did not change the concentration-response curve. 1-Phenylephrine and B-HT933 induced both potent and strong contractions indicating the presence of both alpha 1- and alpha 2-adrenoceptors in these arteries. Although beta-adrenoceptors have been identified in various parts of the ocular circulation by radio-ligand binding techniques our data show that beta-adrenoceptors linked to regulation of vascular tone are only found in intraocular branches of the intraocular segment of long posterior ciliary artery in bovine eyes.(ABSTRACT TRUNCATED AT 400 WORDS)

Nonadrenergic noncholinergic vasodilation in bovine ciliary artery involves CGRP and neurogenic nitric oxide.

PURPOSE: Characterization of the nonadrenergic noncholinergic (NANC) vasodilator innervation in the anterior segment in the bovine eye. METHODS: The neurogenic tetrodotoxin-sensitive response to electrical field stimulation (EFS) of the intraocular segment of the bovine long posterior ciliary artery supplying the ciliary body was recorded using isolated ring segments of this artery mounted on an isometric myograph. After adrenergic and cholinergic receptor blockade (with phentolamine, propranolol, and atropine), the preconstricted vessels were subjected to EFS by passing constant current pulses (0.3 msec, 35 mA, 0.5 to 32 Hz) between two electrodes on either side of the vessel segments. RESULTS: EFS resulted in 60% relaxation of the active tone in 40 vessels. Treatment with capsaicin reduced the NANC response by 16 +/- 2% (P < 0.001) and inhibition of the NO synthase with 1 x 10(-4) M L-NOARG reduced the NANC response by 83 +/- 10% (P < 0.001). Desensitization of the vessels to substance P had no effect. The CGRP(8-37) fragment (1 x 10(-6) M) in the presence of 1 x 10(-4) M L-NOARG reversibly and competitively inhibited the NANC response. L-arginine partly antagonized the inhibition induced by L-NOARG. About 60% of the L-NOARG-sensitive component of the NANC response was inhibited by methylene blue. Combined incubation with capsaicin and L-NOARG nearly abolished the NANC response. The L-NOARG-sensitive/capsaicin-resistant relaxation was present in endothelium denuded vessels. The responses to EFS were blocked by TTX. CONCLUSIONS: The neurogenic NANC vasodilator response in the intraocular part of the bovine long posterior ciliary artery supplying the ciliary body is endothelium independent and consists of two components: a capsaicin-sensitive component mediated by CGRP released from sensory nerve endings and a larger L-NOARG sensitive component mediated by a direct "nitroxidergic" neurotransmission. The size of the nitroxidergic NANC response indicates that it has a physiological relevance in vivo.

Characterization of the CGRP receptor and mechanisms of action in rat mesenteric small arteries.

Rat alpha-calcitonin gene-related peptide-induced concentration-dependent (100 pM-10 nM) relaxations in rat mesenteric small arteries (i.d. approximately 220 microns) contracted with noradrenaline, prostaglandin F2 alpha or K+, however, the maximal relaxation depended on the precontractile stimulus, being highest (95%) in arteries contracted with PGF2 alpha and lowest (51%) in arteries contracted with 125 mM K+. The relaxation was inhibited between 10 pM and 1 nM by removal of the endothelium, but was not antagonized by glibenclamide (1 microM), tetraethylammonium (30 mM), apamine (0.3 microM) and 4-aminopyridine (3 mM). The concentration-response curve to rat alpha-CGRP and human beta-CGRP was shifted to the right in the presence of 1 microM human alpha-CGRP(8-37) indicating a receptor affinity, -log(KB[M]), equal to 7.2 and 7.0, respectively. It is concluded that the relaxation induced by CGRP depends minimally on the endothelium and K(+)-channel opening is not a principal process in the relaxing effect of CGRP, thus a third mechanism must mediate the relaxation in these vessels. The main CGRP receptor type mediating relaxation in rat mesenteric small arteries belongs to the CGRP1 subtype.

Effects of (+)-S-12967 and (-)-S-12968, two enantiomers of a new slow-acting 1,4-dihydropyridine, on rat coronary resistance arteries.

The action of (+)-S-12967 and (-)-S-12968, two isomers of a new 1,4-dihydropyridine molecule (2-(-7-amino-2,5-dioxaheptyl)-3-ethoxycarbonyl-4-(2,3-dichlorop hen yl)-5-methoxycarbonyl-6-methyl 1,4-dihydropyridine), was studied on responses of rat isolated coronary resistance arteries (i.d. about 230 microns) to K+, Ca2+, and 5-hydroxytryptamine (5-HT). Both isomers slowly relaxed coronary arteries contracted with 125 mM K+, reaching a maximal effect in about 2 h. In contrast, the maximal relaxing effect of nifedipine was obtained within 20 min. The response to 125 mM K+ did not recover within the 2-h washout period in vessels exposed to the isomers but returned to pre-drug levels within 40 min in vessels exposed to nifedipine. Nifedipine was 4 times more potent than the (-)-isomer which again was about 200 times more potent that the (+)-isomer. The IC50[M] values were approximately 1 nM, 4 nM and 0.8 microM, respectively. The relaxing effect of the isomers, which has a pKa of 8.6, was dependent on the extracellular pH being greater at high than low pH. Both isomers antagonized the vessel responses to K+ and Ca2+ and 5-HT. Higher concentrations of the isomers were required to antagonize responses to K+ and 5-HT than to Ca2+, probably due to the more depolarized state of the vascular smooth muscle in the latter experiments. In conclusion, the results demonstrate extracellular pH dependence as well as stereoselectivity regarding potency of (+)-S-12967 and (-)-S-12968 in rat coronary arteries.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of induced hypercholesterolemia in rabbits on functional responses of isolated large proximal and small distal coronary arteries.

We studied the effects of hypercholesterolemia on the vascular responses of proximal and distal parts of the rabbit coronary circulation in two consecutive studies. For 12 weeks, New Zealand White rabbits were fed a control diet or a diet with 1% cholesterol dissolved in either 3% coconut oil (study A) or ether (study B). Isolated proximal epicardial and distal intramyocardial coronary arteries from control and hypercholesterolemic rabbits were mounted for isometric tension recording in a double myograph. In study A for hypercholesterolemic rabbits (n = 12), the maximal relaxation and sensitivity to acetylcholine (ACh) were significantly decreased in proximal coronary segments contracted with 30 mmol/l potassium solution compared with segments from control rabbits (n = 13). The only change observed in distal coronary segments was a slight decrease in relaxation in response to low ACh concentrations (10(-8) and 3 x 10(-8) mol/l). However, in study B for proximal coronary and distal coronary segments from hypercholesterolemic rabbits (n = 13), the area under the ACh relaxation curve was increased compared with that of control rabbits (n = 12). Other parameters that were similarly affected in studies A and B include the following: 1) proximal coronary segments from hypercholesterolemic rabbits were more sensitive to sodium nitroprusside (SNP) than were those from control rabbits, but this was not true for distal coronary segments; 2) endothelial removal from arterial segments of control rabbits induced a significant increase in sensitivity and maximal relaxation to SNP of proximal coronary and distal coronary arteries; 3) in segments from hypercholesterolemic rabbits, the absence of endothelium did not alter the response of proximal coronary segments to SNP but did augment the relaxation of distal coronary segments to SNP; 4) the maximal response to 5-hydroxytryptamine in proximal coronary arteries from hypercholesterolemic rabbits was increased compared with those from control rabbits, whereas such changes were not observed in distal coronary arteries; and 5) histological examination showed the presence of atheromatous plaques in proximal coronary but not in distal coronary segments from treated animals. In conclusion, the present investigation demonstrates that induced hypercholesterolemia alters both the structure and function of proximal parts of the coronary circulation. In distal coronary arteries of hypercholesterolemic rabbits, the only change observed was an impaired endothelium-dependent cholinergic relaxation, but even this change appeared to be dependent on the manner in which cholesterol was added to the diet, although parallel studies are required to confirm this.

Difference between aortic and renal vascular reactivity in cyclosporin A treated rats and the effect of cicletanine.

4 Groups of 2 month-old male Wistar rats were treated with a) cyclosporin A (CyA) 30 mg/kg/day alone, b) CyA plus cicletanine (Cic) 60 mg/kg/day, c) vehicle (vegetable oil) 1 ml/100 g rat/day and d) no treatment for 8 weeks. The reactivity of isolated papillary muscle to isoprenaline and Ca2+ was not altered in any of the treated groups. Endothelium-dependent relaxation induced by acetylcholine was inhibited in aorta ring segments from CyA-treated rats as compared to that of control and CyA + Cic-treated rats. The relaxation induced by acetylcholine in rat aortas was similar in all groups in the presence of 10 microM indomethacin. Noradrenaline sensitivity of aortic segments was not affected by any treatments applied. The Ca(2+)-concentration response curves of aorta segments from CyA-treated and CyA + Cic-treated rats were shifted to the right as compared to control rats. In interlobar renal arteries the endothelium-dependent relaxation induced by acetylcholine was not affected by any form of treatment. In renal arteries 10 microM indomethacin increased the maximal relaxation induced by acetylcholine about 50%. In these vessels noradrenaline sensitivity in CyA and CyA + Cic treated rats was higher than in controls. Cocaine, 3 microM, shifted the noradrenaline concentration response curve to the left about 0.4 log units in all renal vessel groups, thus renal vascular smooth muscle sensitivity to noradrenaline was significantly greater in vessels from rats receiving CyA than in vessels from control rats. Administration of CyA induced only slight renal morphological changes. Cic was without effect on CyA induced morphological abnormalities.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcitonin gene-related peptide is a potent vasodilator of bovine retinal arteries in vitro.

Calcitonin gene-related peptide (CGRP) invariably induced a slow acting but potent relaxation of bovine retinal small arteries contracted with PGF2 alpha. Maximal relaxation obtained was 93% and 96% with a pD2-value of 8.97 and 8.86 for rat and human CGRP, respectively; thus the bovine retinal arteries cannot discriminate between CGRP from these two species. The CGRP-induced relaxation was reversible. Substance P was without effect on retinal arteries contracted with PGF2 alpha. Bradykinin relaxed 4 of 18 vessels tested in the concentration range of 11(-11)-10(-8) M whereas the vessels were contracted again at 3 x 10(-8) M. Bradykinin was without effect in the remaining 14 vessels. None of the peptides had a contractile effect on retinal arteries kept relaxed in normal buffer solution. Capsaicin 3 x 10(-5) M induced a relaxation comparable to that obtained by 10(-9) M of CGRP. The capsaicin-induced relaxation was reproducible and it was concentration dependently inhibited by ruthenium red which suggests that capsaicin releases CGRP in the arterial wall. The results indicate that CGRP has a powerful relaxing effect on the retinal vasculature indicating a role for CGRP in ocular blood flow regulation.