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Glucagon-like peptide (GLP)-1 analogs have been implicated as a risk factor for pancreatitis in humans. We investigated whether liraglutide, the once-daily human GLP-1 analog, induces pancreatitis in rats, mice, and monkeys. Pancreata from mice, rats, and nonhuman primates were examined macro- and microscopically. Evaluation of preneoplastic proliferative lesions in the pancreata from nonhuman primates was performed. After 2 years of treatment, 3 of 79 male mice in the control group and 2, 1, 1, and 1 mice in the different liraglutide groups (of 67-79 mice per group) had pancreatitis based on microscopic criteria. For females, the numbers were 0 of 79 mice in the control group and 3 mice in all the liraglutide groups (of 66-76 mice per group). Pancreatitis was not the cause of death in any animals. There were no cases of pancreatitis, macroscopically or microscopically, in 400 rats. Neither pancreatitis nor preneoplastic proliferative lesions was found in monkeys dosed for 87 weeks, with plasma liraglutide exposure 60-fold higher than that observed in humans at the maximal clinical dose. In conclusion, liraglutide did not induce pancreatitis in mice, rats, or monkeys when dosed for up to 2 years and at exposure levels up to 60 times higher than in humans. [ABSTRACT FROM AUTHOR]

Increased pancreas mass and glucagon-positive adenomas have been suggested to be a risk associated with sitagliptin or exenatide therapy in humans. Novo Nordisk has conducted extensive toxicology studies, including data on pancreas weight and histology, in Cynomolgus monkeys dosed with two different human glucagon-like peptide-1 (GLP-1) receptor agonists. In a 52-week study with liraglutide, a dose-related increase in absolute pancreas weight was observed in female monkeys only. Such dose-related increase was not found in studies of 4, 13, or 87 weeks' duration. No treatment-related histopathological abnormalities were observed in any of the studies. Quantitative histology of the pancreas from the 52-week study showed an increase in the exocrine cell mass in liraglutide-dosed animals, with normal composition of endocrine and exocrine cellular compartments. Proliferation rate of the exocrine tissue was low and comparable between groups. Endocrine cell mass and proliferation rates were unaltered by liraglutide treatment. Semaglutide showed no increase in pancreas weight and no treatment-related histopathological findings in the pancreas after 13 or 52 weeks' dosing. Overall, results in 138 nonhuman primates showed no histopathological changes in the pancreas associated with liraglutide or semaglutide, two structurally different GLP-1 receptor agonists., (© 2014 by the American Diabetes Association.)

The aim of the present study was to examine the calcium activity of C8-T5 dorsal root ganglion (DRG) neurons from Zucker diabetic fatty rats. In total, 8 diabetic ZDF fatty animals and 8 age-matched control ZDF lean rats were employed in the study. C8-T5 dorsal root ganglia were isolated bilaterally from 14 to 18 weeks old rats, and a primary culture was prepared. Calcium activity was measured ratiometrically using the fluorescent Ca(2+)-indicator Fura-2 acetoxymethyl ester. All neurons were stimulated twice with 20 mM K(+), followed by stimulation with either 0.3 or 0.5  μ M Capsaicin, alone or in combination with algogenic chemicals (bradykinin, serotonin, prostaglandin E2 (all 10(-5) M), and adenosine (10(-3) M)) at pH 7.4 and 6.0. Neurons from diabetic animals exhibited an overall increased response to stimulation with 20 mM K(+) compared to neurons from control. Stimulation with Capsaicin alone caused an augmented response in neurons from diabetic animals compared to control animals. When stimulated with a combination of Capsaicin and algogenic chemicals, no differences between the two groups of neurons were measured, neither at pH 7.4 nor 6.0. In conclusion, diabetes-induced alterations in calcium activity of the DRG neurons were found, potentially indicating altered neuronal responses during myocardial ischemia.

Liraglutide and exenatide are glucagon-like peptide receptor (GLP-1R) agonists used in the treatment of type 2 diabetes. Both molecules have been associated with the development of thyroid C-cell tumors after lifetime exposure in rodents. Previously, it has been reported that these tumors are preceded by increased plasma calcitonin and C-cell hyperplasia. We can now document that the murine C-cell effects are mediated via GLP-1R. Thus, 13 wk of continuous exposure to GLP-1R agonists was associated with marked increases in plasma calcitonin and in the incidence of C-cell hyperplasia in wild-type mice. In contrast, similar effects were not seen in GLP-1R knockout mice. Human C-cell cancer is often caused by activating mutations in the rearranged-during-transfection (RET) protooncogene. We developed an immunohistochemical method to assess RET activation in tissues. Liraglutide dosing to mice was not found to activate RET. Further evaluation of the signaling pathways demonstrated that liraglutide increased ribosomal S6, but not MAPK kinase, phosphorylation. These observations are consistent with effects of GLP-1R agonists on rodent C cells being mediated via mammalian target of rapamycin activation in a RET- and MAPK-independent manner.

The influence of ageing on the passive and active tension and pharmacodynamic characteristics of intramural coronary arteries from 3-month-old and 2-year-old male Wistar rats was investigated using an isometric myograph. The passive vessel wall tension measured in Ca(2+)-free physiological salt solution at L(0) was significantly greater in arteries from old rats (1.46 ± 0.10 Nm(-1), n = 7) than in young rats (1.13 ± 0.13 Nm(-1), n = 6). However, the maximal active tension at L(0) was similar. The spontaneous myogenic tone was increased by age and the vasorelaxation induced by extracellular K(+) was significantly higher in coronary arteries of old rats. The sensitivity (pD(2)) to 5-HT was significantly higher in arteries from old (6.43 ± 0.11, n = 22) than from young rats (6.16 ± 0.08, n = 29). Ketanserin induced a concentration-dependent rightward shift of the 5-HT concentration-response curve in arteries from both young and old rats. The slopes of the regression lines of the Schild plots were not significantly different from unity and the estimated pK(B) values for ketanserin were similar. In conclusion, ageing is associated with changes in passive mechanical characteristics as well as changes in pharmacological properties in rat coronary small arteries., (Copyright © 2012 S. Karger AG, Basel.)

The aim of the present study was to examine spinal processing of cardiac and somatic nociceptive input in rats with STZ-induced diabetes. Type 1 diabetes was induced with streptozotocin (50mg/kg) in 14 male Sprague-Dawley rats and citrate buffer was injected in 14 control rats. After 4-11 weeks, the rats were anesthetized with pentobarbital, ventilated and paralyzed. A laminectomy enabled extracellular recording of T(3) spinal cord neuronal activity. Intrapericardial administration of a mixture of algogenic chemicals (bradykinin, serotonin, prostaglandin E(2) (all at 10(-5)M), and adenosine (10(-3)M)) was applied to activate nociceptors of cardiac afferent nerve endings. Furthermore, somatic receptive properties were examined by applying innocuous (brush and light pressure) and noxious (pinch) cutaneous mechanical stimuli. Diabetes-induced increases in spontaneous activity were observed in subsets of neurons exhibiting long-lasting excitatory responses to administration of the algogenic mixture. Algogenic chemicals altered activity of a larger proportion of neurons from diabetic animals (73/111) than control animals (55/115, P<0.05). Some subtypes of neurons exhibiting long-lasting excitatory responses, elicited prolonged duration and others, had a shortened latency. Some neurons exhibiting short-lasting excitatory responses in diabetic animals elicited a shorter latency and some a decreased excitatory change. The size of the somatic receptive field was increased for cardiosomatic neurons from diabetic animals. Cutaneous somatic mechanical stimulation caused spinal neurons to respond with a mixture of hyper- and hypoexcitability. In conclusion, diabetes induced changes in the spinal processing of cardiac input and these might contribute to cardiovascular autonomic neuropathy in patients with diabetes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The influence of ageing and endothelium removal on the sensitivity and contractile response of rat coronary arteries to intracellular Ca2+ ([Ca2+]i) during activation with serotonin (5-HT) and membrane depolarisation with 125 mM K+ was investigated. The sensitivity and contractile response of coronary arteries to 5-HT were significantly higher in 2-year-old than in 3-month-old rats. The receptor responsible for the 5-HT-induced contractions in coronary arteries belongs to a population of 5-HT2 receptors in both young and old rats based on the Schild plot. The resting levels of [Ca2+]i and active tension were both increased by age and endothelium removal. During depolarisation with 125 mM K+, the sensitivity to [Ca2+]i and maximal tension induced by [Ca2+]i were not affected by age or endothelium. During activation with 10 microM 5-HT, the maximal tension induced by [Ca2+]i was increased by age but not affected by endothelium, whereas the sensitivity to [Ca2+]i was increased by endothelium removal. In conclusion, ageing is associated with an increased sensitivity to 5-HT in rat coronary small arteries. The increased sensitivity to 5-HT seems to involve an augmented contractile response to [Ca 2+]i in 5-HT-activated coronary arteries and a diminished endothelial basal vasodilator function.

A new method for determination of first-order elimination constants for dipeptides is presented. The peptides are hydrolysed by plasma enzymes into amino acids, and ortho-phthaldialdehyde (OPA) is used to react with free primary amino groups. The concentration of free amino groups can, thus, be followed using simple spectrophotometry. A mathematical model for the concentration of free primary amino groups with time is presented through which the elimination constant, and thus the half life, can be determined by curve fitting. The method is applied to inhibitors of angiotensin-converting enzyme derived from the primary structure of milk proteins. The results show that these dipeptides have in vitro half lives ranging from 4.3-64 min, when incubated with 50% rat plasma. This explains why these casokinins in vivo only cause a very moderate and short-lasting inhibition. The model for calculation of elimination constant is limited to dipeptides that do not contain a C-terminal proline. The derivatization method can be applied to longer peptides as a crude indicator of peptide hydrolysis, but does not allow calculation of their elimination constants per se.

We investigated the type of desensitization of calcitonin gene-related peptide (CGRP)-induced responses in rat isolated intramural coronary arteries using isometric myograph and FURA-2 technique. In coronary arteries precontracted with 9,11-dideoxy-11alpha,9alpha-epoxymethanoprostaglandin F2alpha (U46619), development of tachyphylaxis to CGRP is characterized by significant attenuation of CGRP-induced maximal reduction in the tension and [Ca2+](i) during the second CGRP concentration-response curve; however, there was no further reduction in the CGRP-induced maximum relaxation during the third CGRP concentration-response curve. There was no sign of tachyphylaxis to CGRP when CGRP concentration-response curves were recorded in 36 mM K+-depolarized coronary arteries contrary to the results obtained in 300 nM U46619-precontracted coronary arteries. Preincubation with colchicine did not prevent the development of tachyphylaxis to CGRP in U46619-precontracted coronary arteries, indicating no role for endocytosis. Development of tachyphylaxis to CGRP was completely abolished by preincubating the coronary arteries with 1 microM RO 31-8220, indicating a role for protein kinases. Pre-exposure of the coronary arteries to isoprenaline or forskolin did not attenuate the CGRP-induced relaxation in these vessels, indicating that the cAMP-protein kinase A (PKA) pathway is not involved. Like CGRP, the coronary arteries developed tachyphylaxis toward isoprenaline during the second exposure. However, there was no sign of tachyphylaxis to either forskolin or dibutyryl cAMP (dbcAMP) during the second exposure. In conclusion, these results suggest that development of tachyphylaxis to CGRP in U46619-precontracted coronary is related to CGRP receptor-mediated activation of protein kinase.

Inhibition of angiotensin converting enzyme (ACE) has been observed with a variety of different peptides, and peptide fragments with inhibitory capabilities have been identified within many different proteins, including milk proteins. The purpose of this study therefore was to identify new short peptides with inhibitory properties from the primary structure of milk proteins and to characterize them in vitro and in vivo, since no milk derived ACE inhibitors have previously been evaluated for their ability to inhibit ACE in vivo. In vitro, 8 of 9 dipeptides were found to be competitive inhibitors of ACE. The IC50 was significantly lower when an angiotensin I-like substrate was used, than when a bradykinin-like substrate was used. Using three different in vivo models for ACE inhibition, a very moderate effect was observed for three of the new peptides, but only for up to 6 or 12 minutes. Nothing was observed with two reference compounds that are reported to be hypotensive ACE-inhibitors derived from milk proteins. This raises the question whether the mechanism of hypotensive action is straightforward inhibition of ACE in vivo.

A total of 26 strains of wild-type lactic acid bacteria, mainly belonging to Lactococcus lactis and Lactobacillus helveticus, were assayed in vitro for their ability to produce a milk fermentate with inhibitory activity towards angiotensin converting enzyme (ACE). It was clear that the test strains in this study, in general, produce inhibitory substances in varying amounts. Using a spectrophotometric assay based on amino group derivatization with ortho-phthaldialdehyde as a measure of relative peptide content, it was shown that there is a significant correlation between peptide formation and ACE inhibition, indicating that peptide measurement constitutes a convenient selection method. The effect of active fermentates on in vivo ACE activity was demonstrated in normotensive rats. The pressor effect of angiotensin I (0.3 microg/kg) upon intravenous injection was significantly lower when rats were pre-fed with milks fermented using two strains of Lactobacillus helveticus. An increased response to bradykinin (10 microg/kg, intravenously injected) was observed using one of these fermented milks. It is concluded that Lactobacillus helveticus produces substances which in vivo can give rise to an inhibition of ACE. The inhibition in vivo was low compared to what can be achieved with classical ACE inhibitors. The clinical relevance of this finding is discussed. This work is the first in which an effect of fermented milk on ACE in vivo has been demonstrated, measured as decreased ability to convert angiotensin I to angiotensin II.

In this study, two strains of Lactobacillus helveticus were used to produce fermented milk rich in angiotensin-converting enzyme (ACE) inhibitors. In vitro tests revealed that the two milks contained competitive inhibitors of ACE in amounts comparable to what has been obtained in previously reported studies. The two milks were administered by gavage to spontaneously hypertensive rats that had had a permanent aortic catheter inserted through the left arteria carotis, and mean arterial blood pressure and heart rate were monitored from 4 to 8 h after administration. Unfermented milk and milk fermented with a lactococcal strain that does not produce inhibitors were used as controls. Highly significant blood pressure effects were observed; i.e., milk fermented with the two strains of L. helveticus gave a more pronounced drop in blood pressure than the controls. Significant differences in heart rate effects were detected with one of the strains.

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.

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.

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.

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.

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.

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+.

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.

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.

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.

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.

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.

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 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)

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.

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.

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)

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)

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.

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.

1. The actions of (+)-S-12967 and (-)-S-12968 two isomers of a new 1,4-dihydropyridine (DHP) derivative, were studied on 125 mM K(+)-, Ca(2+)- and noradrenaline-induced contractions in rat isolated mesenteric resistance arteries and compared to those of nifedipine. 2. The action of (+)-S-12967 and (-)-S-12968 was slow in onset in contrast to nifedipine. Both isomers had a dual contractile and relaxant action in arteries contracted with 125 mM K+; however, the (-)-isomer was about 300 times more potent than the (+)-isomer. The response to 125 mM K+, being depressed by 70%, recovered within 20 to 30 min for all DHP derivatives. All vessels were treated with 1 x 10(-6) M phenoxybenzamine thus excluding the possibility that the contraction is mediated by activation of amine-receptors. 3. Both (+)-S-12967 and (-)-S-12968 at low concentrations potentiated responses induced by Ca2+ in arteries activated by 125 mM K+ and inhibited the responses at higher concentrations. (+)-S-12967 and (-)-S-12968 had no contractile action in arteries kept in normal buffer. Nifedipine had only an inhibitory action on vessel responses to 125 mM K+ and Ca2+. 4. Both isomers and nifedipine depressed the maximal vessel response to noradrenaline by about 20% and 44%, respectively. 5. The results confirm that DHP calcium antagonists selectively inhibit vascular smooth muscle responses induced by high potassium and that the potency of 1,4-DHP isomers may vary considerably. Furthermore, since the agonistic/antagonistic properties on the calcium channel were shared by both stereoisomers of the 1,4-DHP molecule and apparently dependent on their concentration and the vascular smooth muscle membrane potential, it suggests that the agonistic action of 1,4-DHPs may be ascribed to functional characteristics of their binding site regulating the Ca2l -channel.

1. The effects of rat- and human-CGRP and capsaicin were studied in isolated rings of rat proximal epicardial (PC) and distal intramyocardial (DC) coronary arteries. 2. The relaxing effect of rat-CGRP was dependent on the level of vessel tone induced by prostaglandin F2 alpha (PGF2 alpha) in PC but not in DC arteries. Submaximally contracted DC and PC arteries were more sensitive to rat- than human-CGRP. There was no difference in sensitivity to rat- and human-CGRP between PC and DC arteries. 3. Substance P elicited a small relaxation only in 4 of the 6 PC arteries tested. PC and DC arteries were concentration-dependently relaxed by capsaicin. The relaxation was partly inhibited by ruthenium red, thus suggesting that capsaicin causes specific release of CGRP from sensory nerve endings in rat coronary arteries. 4. The relaxant effect of rat-CGRP was antagonized by endothelium removal and indomethacin but not methylene blue in endothelium-intact PC arteries. The relaxation in DC arteries was not affected by any of these treatments, indicating a heterogeneous involvement of the endothelium in CGRP-mediated coronary vasodilatation and the release of a cyclo-oxygenase product in PC arteries in rats. 5. Glibenclamide had no inhibitory effect on the CGRP-induced relaxation of PC and DC arteries, thus excluding the involvement of glibenclamide-sensitive K(+)-channels in the mechanism of action of CGRP in rat coronary arteries.

Acetylcholine induced a variable concentration-dependent relaxation of bovine isolated retinal small arteries contracted with PGF2 alpha. The acetylcholine-mediated relaxation was linearly related to the sodium nitroprusside-induced relaxation, suggesting that the endothelium is well preserved in the vessels and that the variable effect of acetylcholine is due to variations in the soluble guanylate-cyclase enzyme activity in the smooth muscle. The vessels became desensitized to acetylcholine by repeated exposures. L-arginine and indomethacin did not abolish the desensitization. The vessels also became desensitized to the direct smooth muscle relaxing effect of sodium nitroprusside, indicating that desensitization to the endothelium-dependent relaxation by acetylcholine is related primarily to the vascular smooth muscle cells. Atropine, methylene blue and removal of endothelium abolished the acetylcholine-induced relaxation completely, whereas indomethacin had no inhibitory action on the acetylcholine-induced relaxation, suggesting that acetylcholine mediates release of EDRF (nitric oxide: NO) through stimulation of muscarinic receptors. Methylene blue contracted endothelium intact retinal arteries but a spontaneous tone was not present in endothelial denuded arteries. This may indicate a basal release of both a contractile factor, e.g. endothelin, and a relaxing factor. NO, form the retinal endothelium. The results demonstrate that endothelial-derived factors may participate in normal as well as pathophysiological regulation of retinal vascular smooth muscle tone.

1. Neuropeptide Y (NPY) induced a concentration-dependent contraction of isolated rings of proximal epicardial (PC) and distal intramural (DC) coronary arteries of the rat, with an EC50 of ca. 1 x 10(-7) M. The NPY-induced contraction at 3 x 10(-7) M was significantly smaller in PC than DC arteries: 34% vs. 55% of the 125 mM K(+)-induced response, respectively. 2. NPY (2 x 10(-8) M) increased the sensitivity to noradrenaline (NA) and 5-hydroxytryptamine (5-HT) more in PC (4.2 and 2.8 fold, respectively) than in DC arteries (2.2 and 1.4 fold, respectively). The maximal contractile response to NA and 5-HT was increased more in DC (43% and 29%, respectively) than in PC arteries (20% and 12%, respectively). 3. Removal of the endothelium increased the sensitivity and maximal response to NPY as well as the spontaneous myogenic tone in PC but not in DC arteries. NPY had no relaxing effect on PC and DC arteries submaximally contracted with 10(-6) M prostaglandin F2 alpha, suggesting that spontaneous rather than stimulated release of endothelium-derived relaxing factor (EDRF) depresses the contractile action of NPY in PC arteries. 4. The results indicate a heterogeneity in the contractile and potentiating action of NPY in rat coronary arteries depending on size or location in the coronary circulation.

1. The 5-hydroxytryptamine (5-HT)-receptor subtype and affinity for 5-HT was determined in large and small coronary arteries isolated as ring segments from the proximal and distal part of the left coronary artery of 3 month (young) and 2-year old (old) rats. 2. Ketanserin induced a rightward shift of the 5-HT concentration-response curve in both proximal and distal coronary arteries from young rats. The slopes of the Schild-plots were indistinguishable from unity and the estimated pA2 values were 9.11 and 9.27 for proximal and distal coronary arteries, respectively. These data indicate a homogeneous population of 5-HT2-like receptors in the coronary arteries. 3. The contractile effect of 5-HT as well as the sensitivity to 5-HT was greater in proximal and distal coronary arteries from old than from young rats. 4. The apparent 5-HT2-receptor affinity, -log(KA[M]), and fractional receptor-occupancy for relative responses between 10% and 90% of maximum was determined by partial irreversible inhibition of the 5-HT2-receptors with phenoxybenzamine. 5. Ageing was associated with an increase in 5-HT2-receptor affinity for 5-HT in both proximal and distal coronary arteries, whereas the fractional receptor occupancy for half-maximal response to 5-HT decreased with age. 6. 5-HT2-receptor affinity for 5-HT could account for the 5-HT sensitivity of distal coronary arteries in both young and old rats but not in proximal coronary arteries as the slope of the regression line of plots of 5-HT2-receptor affinity vs. sensitivity was indistinguishable from unity in only the distal vessels. 7. The 5-HT2-receptor affinity for 5-HT was linearly correlated to the fractional receptor occupancy for half maximal response, suggesting that the 5-HT2-receptor reserve or density down-regulates the receptor affinity for 5-HT. 8. The results indicate that the increase in 5-HT sensitivity and contractile effect in rat coronary arteries rely upon an increase in both 5-HT2-receptor agonist affinity and efficiency of the excitation-contraction coupling process in the vascular smooth muscle.

Endothelin-1 (porcine/human) induced a prompt, reversible, potent, concentration-dependent contraction of bovine isolated retinal small arteries (internal diameter, approximately 200 microns) with a concentration of endothelin-1 required to give half-maximal contraction of 2 x 10(-10) M. The maximal contraction induced by endothelin-1 was equal to 89% of the maximal contractile capacity. The vessel response to endothelin-1 was dependent on extracellular Ca2+. Withdrawal of extracellular Ca2+ or addition of nitrendipine, 10(-6) M, reduced the response of vessels contracted with 10(-9) M endothelin-1 by 80% and 75%, respectively. These results indicate that the endothelin-1-induced contraction of retinal arteries is dependent on an influx of extracellular Ca2+ through membrane potential-operated calcium channels. Endothelin-1, 10(-13)-10(-10) M, did not induce a relaxation of endothelium-intact arteries, indicating that endothelin-1 is incapable of releasing endothelium-derived relaxing factor in the retinal circulation. These results suggest that endothelin may participate in the regulation of retinal artery tone.

Histamine relaxed PGF2 alpha-precontracted rat isolated middle cerebral arteries (ID approximately 230 microns) concentration dependently with a pD2 of 5.31 (EC50: 5 x 10(-6) M). Cimetidine induced a concentration-dependent rightward shift of the histamine concentration-response curve of endothelium-intact arteries. The slope of the Schild plot was indistinguishable from unity, and the estimated pA2 for cimetidine was 6.14. The selective H2-receptor agonist dimaprit induced a concentration dependent relaxation of the cerebral arteries similar to that induced by histamine. This indicates that the histamine receptor mediating the relaxation in rat middle cerebral arteries belongs to the H2-receptor subtype. 2-Pyridylethylamine, a selective H1-receptor agonist, induced a small concentration-dependent contraction of the arteries with a pD2 of 4.16. Mepyramine, a selective H1-receptor antagonist had no potentiating effect on the relaxation induced by histamine, suggesting either that the contractile effect of 2-pyridylethylamine is nonselective or that H1 receptors mediating contraction are of minor importance for the overall histamine response. The selective H3-receptor agonist, (R)-alpha-methylhistamine, was without effect in a specific concentration range (10(-7)-10(-5) M) excluding participation of H3 receptors in the histamine-induced relaxation of these vessels. Indomethacin did not affect the vessel response to histamine, but removal of the endothelium and treatment of endothelium-intact arteries with 3 x 10(-6) M methylene blue induced a similar 0.5 log rightward shift of the histamine concentration-response curve.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of histamine on bovine isolated retinal small arteries (internal diameter, approximately 240 microns) was studied. Histamine induced a concentration-dependent relaxation in 43 of 53 vessels. The histamine-induced relaxation involves primarily activation of H1-receptors, with H2-receptors also affected as evidenced by the effect of selective histamine-receptor agonists and antagonists. The histamine-induced relaxation was dependent on the endothelium and seem to involve release of both endothelium-derived relaxing factor (EDRF) and a product which was inhibited by indomethacin, probably prostaglandin I2 (prostacyclin). The development of tachyphylaxis to the action of histamine seemed to rely on desensitization of the vascular smooth muscle cells to the relaxing effect of EDRF.

The spontaneous myogenic activity of rings of human posterior ciliary arteries was studied in vitro. All arteries from six patients had an intrinsic myogenic tone, which was dependent on extracellular calcium and inversely correlated with age. The myogenic tone was stable without rhythmic variations, except in one artery. The level of spontaneous myogenic tone was higher in vessels where the endothelium was removed than in normal arteries. Acetylcholine induced a complete and concentration dependent relaxation of endothelium-intact but not endothelium-denuded arteries. Indomethacin induced a small concentration dependent contraction of the arteries. Methylene blue did also contract the arteries concentration dependently. The experiments indicate that the myogenic tone of human posterior ciliary arteries is regulated by prostaglandin formation (prostacyclin?) as well as release of endothelium-derived relaxing factor in the arteries. The results indicate that the blood flow regulatory capacity of these arteries may be endangered by aging and endothelial damage, which could increase the probability of developing, e.g. ischemic damage of the optic nerve head.

The effect of thrombin was tested in vitro on rings of bovine retinal small arteries (internal diameter approximately 200 microns). Cumulative addition of thrombin (0.001-10 units/ml) induced a variable concentration-dependent contraction of the retinal arteries. The contractions were slow in onset and reached a plateau after 5-8 minutes when thrombin was added cumulatively to the organ bath. Two vessels remained contracted greater than 1 hour after wash-out of thrombin. Maximum vessel contraction induced by thrombin was equal to 43% of the vessel Emax (1.36 N/m), with an effective concentration at the 50% level of 0.04 units/ml. Vessel contraction induced by 1 unit/ml of thrombin was, in contrast, transient, reaching a maximum within 2-4 minutes. Thereafter the vessel tension declined again almost back to baseline within the next 10-20 minutes. Contractions to thrombin could not be repeated nor could it be elicited with thrombin inactivated with heat or antithrombin-III. Treatment of vessels with 10(-6) M phenoxybenzamine had no effect on the thrombin-induced vessel response. These findings indicate that the contractile effect of thrombin depends on its catalytic activity. Indomethacin at a concentration of 10(-5) M did not affect the thrombin-induced vessel response. Methylene blue at a concentration of 3 X 10(-6) M potentiated the thrombin-induced response in the larger, greater than 200-microns diameter retinal arteries. The ensuing relaxation of the arteries, after maximal tone was reached, was slower than in the control.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of angiotensin-II (A-II) was studied on ring segments of the terminal extraocular branches of the posterior ciliary artery isolated from human enucleated eyes. It induced a potent concentration-dependent contraction on top of the spontaneous myogenic tone of all arteries studied from five patients with the concentration required to give half-maximal response equal to 51 nM. The spontaneous tone and maximal increase in vessel wall tension induced by A-II was equal to 51% of Emax. The relative response and sensitivity to A-II was unchanged in three endothelial denuded vessels, but the spontaneous tone increased. The arteries became completely insensitive to A-II after one exposure. These results show an immediate direct contractile effect of A-II on human posterior ciliary arteries, but the development of pronounced tachyphylaxis indicates that A-II is probably not an important factor in reducing blood flow to the optic nerve head.

Removal of the endothelium from isolated rat proximal and distal coronary artery segments shifted the 5-HT concentration-response curve to the left without affecting, the maximal contractile response. 5-HT had no relaxing effect in 10(-5) M prostaglandin F2 alpha-precontracted vessels with an intact endothelium in the presence of 10(-5) M ketanserin. The spontaneous myogenic tone increased in both proximal and distal coronary artery segments after the endothelium had been removed. Indomethacin (10(-5) M) reduced the response of the proximal coronary artery segments to 5-HT by 35% but indomethacin had no effect on the 5-HT concentration-response curve of the distal coronary artery segments. Indomethacin relaxed precontracted (40 mM potassium) proximal coronary artery segments independently of the presence of the endothelium, suggesting a non-specific relaxing effect of indomethacin in these arteries. It is concluded that rat coronary artery endothelium is unresponsive to 5-HT because it lacks 5-HT1 receptors. The increased 5-HT sensitivity and spontaneous myogenic tone of endothelium-denuded rat coronary arteries is probably due to the elimination of the relaxing stimulus mediated by spontaneously released endothelium-derived relaxing factor.

The active and passive wall tension--internal circumference (diameter) relations of isolated ring segments of bovine retinal small arteries (i.d. ca. 200 microns) were studied in vitro using an isometric myograph. The active tension of the vessels (when fully activated with 10(-5) M 5-HT and 10(-5) M PGF2 alpha in high K(+)-saline) reached a maximum (1.2 N m-1) at an internal circumference, L0, where the passive tension (measured in Ca2(+)-free solution) were 0.89 N m-1. Below and above L0 the active tension fell linearly, the zero tension intercepts being 0.48 L0 and 1.61 L0. The passive wall tension rose exponentially as a function of the internal circumference (diameter) and was 43 times higher at 1.4 L0 than at L0. A small transient spontaneous contraction was observed after rapid stretch of the arteries. Papaverine and Ca2(+)-free solution did not change the wall tension of vessels stretched to L0, indicating absence of stable spontaneous myogenic tone in the vessels. Histological examination showed that the retinal artery media on average had three smooth muscle cell layers which in total were 12 microns thick. The media/lumen ratio was 7%. Smooth muscle cell volume was 1228 micron3, constituting about 72% of the media volume. The active wall tension--internal circumference relation and morphology of bovine retinal small arteries is generally equal to that of arteries of similar size from other species, but active force generation of the retinal vascular smooth muscle cells is considerably lower.

1. The effect of noradrenaline (NA) on the vascular smooth muscle and endothelial cells in isolated ring segments from the proximal and distal part of the left coronary artery (LCA) in rats was examined. 2. NA had a weak concentration-dependent contractile effect on proximal but relaxed distal segments of the LCA. The maximal NA-induced contraction of the proximal segments was doubled while the relaxation of the distal LCA segments was converted to a contraction after blockade of beta-adrenoceptors with propranolol 3 x 10(-6) M, thus indicating the presence of both alpha- and beta-adrenoceptors in the arteries, with dominance of alpha-adrenoceptors and of beta-adrenoceptors in the proximal and distal segments of the LCA, respectively. 3. The contractile effect of NA (beta-adrenoceptors blocked) was doubled in the proximal LCA segments after the endothelium was removed. Endothelial denudation had, in contrast, no potentiating effect on the contractile response of the distal arteries to NA. Both proximal and distal segments became more sensitive to the contractile action of NA after removal of the endothelium. 4. The spontaneous myogenic tone increased in both proximal and distal LCAs after endothelial removal, indicating spontaneous release of a relaxing endothelial factor in the vessels. 5. Following contraction with prostaglandin F2 alpha (PGF2 alpha), and in the presence of propranolol, 3 x 10(-6) M, and prazosin, 10(-6) M, NA induced an endothelium-dependent relaxation of only proximal but not distal segments of the precontracted LCA. The NA-induced relaxation of the proximal segments of the LCA was not altered by indomethacin 10- M but was completely abolished after incubation with methylene blue, 3 x 10-6 M, or following endothelium removal. These results are compatible with NA-induced release of EDRF in these arteries. 6. The selective alpha 2-adrenoceptor agonist, B-HT 933, only induced a weak relaxation of PGF2 alpha,-precontracted proximal (endothelium intact) LCA segments at a concentration of 10-4M. The NA-induced relaxation of these vessels was unaffected by incubating the vessels with 10- IM B-HT 933. The NA relaxation response curve was shifted ca 1.1 log unit to the right by rauwolscine, 1o- 6M, giving an estimated pA2-value of 7.12. The receptor through which NA activates the endothelium appears to be of an atypical alpha 2-subtype.

The effect of angiotensin II was studied in vitro on ring segments of bovine retinal resistance arteries (i.d. 126-271 microns) and posterior ciliary arteries (i.d. 207-1153 microns). Although the retinal resistance arteries were responsive to 5-hydroxytryptamine, prostaglandin F2 alpha, and changes in extracellular K(+)-concentration, they did not, in contrast to the posterior ciliary arteries, contract to cumulative or single doses of angiotensin II. In the latter arteries, angiotensin II induced a small concentration dependent contraction, 5% of maximal 125 mM K(+)-induced response, with a pD2-value of 9.3. The single addition of 10(-6) M angiotensin II increased the maximal vessel response of the posterior ciliary arteries three times to angiotensin II. Tachyphylaxis was pronounced in the posterior ciliary arteries, in which the response to angiotensin II could not be repeated. Indomethacin (10(-5) M), methylene blue (3 x 10(-6) M), or removal of endothelium did not make the retinal resistance arteries responsive to angiotensin II. Retinal arteries precontracted with 30 mM potassium did not respond to angiotensin II. Angiotensin II did not potentiate the 5-hydroxytryptamine- and noradrenaline concentration-response characteristics of both retinal resistance and posterior ciliary arteries. Although angiotensin II-receptors have been detected in bovine retinal vascular smooth muscle using radioligand-binding technique, the present results suggest that these receptors are non-functional in respect to regulation of retinal resistance artery tone.

The reactivity of isolated ring segments (i.d. ca. 200 microns) of calf retinal resistance arteries to arachidonic acid derivatives were studied in vitro. Prostaglandin F2 alpha (PGF2 alpha) prostaglandin E2 (PGE2), and a thromboxane A2 analogue, 9,11-dideoxy-9 alpha,11 alpha-epoxymethane-prostaglandin F2 alpha, (TX2 alpha), induced a concentration dependent contraction with the order of potency: Tx2 alpha greater than PGE2 greater than PGF2 alpha with pD2-values (-log[EC50(M)]) of 6.88, 6.19 and 5.20, respectively. The maximal active tension development of the vessels induced by Tx2 alpha, PGE2 and PGF2 alpha, were 1.57 N m(-1), 1.05 N m(-1) and 1.19 N m(-1), corresponding to 94%, 57% and 67% of Emax, respectively. The active pressure development, and estimate of the maximum pressure which the vessels may be able to contract against in vivo, was 18 kPa, 12 kPa and 14 kPa (138, 89 and 110 mmHg) for Tx2 alpha, PGE2 and PGF2 alpha, respectively. Prostaglandin I2 (prostacyclin, PGI2) induced a concentration-dependent relaxation of retinal resistance arteries precontracted with 10(-5) M PGF2 alpha. The maximal relaxation induced by PGI2 amounted to 65% of the control vessel response induced by PGF2 alpha (1.27 N m(-1] with a -log[EC50(M)] value of 6.51. The results indicate that arachidonic acid derivatives are potent and effective agents capable of regulating bovine retinal resistance artery smooth muscle tension in vitro.