Genetic fuzzy system predicting contractile reactivity patterns of small arteries.

Monitoring of physiological surrogate end points in drug development generates dynamic time-domain data reflecting the state of the biological system. Conventional data analysis often reduces the information in these data by extracting specific data points, thereby discarding potentially useful information. We developed a genetic fuzzy system (GFS) algorithm that is capable of learning all information in time-domain physiological data. Data on isometric force development of isolated small arteries were used as a framework for developing and optimizing a GFS. GFS performance was improved by several strategies. Results show that optimized fuzzy systems (OFSs) predict contractile reactivity of arteries accurately. In addition, OFSs identified significant differences that were undetectable using conventional analysis in the responses of arteries between groups. We concluded that OFSs may be used in clustering or classification tasks as aids in the objective identification or prediction of dynamic physiological behavior.

Heart ischaemia-reperfusion induces local up-regulation of vasoconstrictor endothelin ETB receptors in rat coronary arteries downstream of occlusion.

BACKGROUND AND PURPOSE: Endothelins act via two receptor subtypes, ETA and ETB . Under physiological conditions in coronary arteries, ETA receptors expressed in smooth muscle cells mediate vasoconstriction whereas ETB receptors mainly found in endothelial cells mediate vasorelaxation. However, under pathophysiological conditions, ETB receptors may also be expressed in vascular smooth muscle cells mediating vasoconstriction. Here, we have investigated whether vasoconstrictor ETB receptors are up-regulated in coronary arteries after experimental myocardial ischaemia in rats. EXPERIMENTAL APPROACH: Male Sprague-Dawley rats were subjected to either heart ischaemia-reperfusion (15 min ischaemia and 22 h reperfusion), permanent ischaemia (22 h) by ligation of the left anterior descending coronary artery, or sham operation. Using wire myography, the endothelin receptor subtypes mediating vasoconstriction were examined in isolated segments of the left anterior descending and the non-ligated septal coronary arteries. Endothelin receptor-mediated vasoconstriction was examined with cumulative administration of sarafotoxin 6c (ETB receptor agonist) and endothelin-1 (with or without ETA or ETB receptor blockade). The distribution of ETB receptors was localized with immunohistochemistry and quantified by Western blot. KEY RESULTS: Endothelin ETB receptor-mediated vasoconstriction and receptor protein levels were significantly augmented in coronary arteries situated downstream of the occlusion after ischaemia-reperfusion compared with non-ischaemic arteries. In contrast, the ETA receptor-mediated vasoconstriction was unaltered in all groups. CONCLUSIONS AND IMPLICATIONS: Ischaemia-reperfusion induced local up-regulation of ETB receptors in the smooth muscle cells of coronary arteries in the post-ischaemic area. In contrast, in non-ischaemic areas, ETB receptor function was unaltered.

Differential localization and characterization of functional calcitonin gene-related peptide receptors in human subcutaneous arteries.

AIM: Calcitonin gene-related peptide (CGRP) and its receptor are widely distributed within the circulation and the mechanism behind its vasodilation not only differs from one animal species to another but is also dependent on the type and size of vessel. The present study examines the nature of CGRP-induced vasodilation, characteristics of the CGRP receptor antagonist telcagepant and localization of the key components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) of the CGRP receptor in human subcutaneous arteries. METHODS: CGRP-induced vasodilation and receptor localization in human subcutaneous arteries were studied by wire myograph in the presence and absence of the CGRP receptor antagonist telcagepant and immunohistochemistry respectively. RESULTS: At concentrations of 1, 3, 5, 10 and 30 nm, telcagepant had a competitive antagonist-like behaviour characterized by a parallel rightwards shift in the log CGRP concentration-tension/calcium curve with no depression of the maximal relaxation. CGRP-induced vasodilation was not affected by mechanical removal of the endothelium or addition of L-NG-nitroarginine methyl ester and indomethacin, antagonists for synthesis of nitric oxide and prostaglandins, respectively. CLR and RAMP1 were localized in the vascular smooth muscle and endothelial cells. CONCLUSION: The present results indicate that CGRP exerts its vasodilatory effect in human subcutaneous arteries by binding to its receptors located on the smooth muscle cells and is suggested to be endothelium-independent. In conclusion, these results underline the dynamic distribution of CGRP receptor components in the human circulation reflecting the important role of CGRP in fine tuning of the blood flow in resistance arteries.

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.

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.

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.