.
`(cid:14)
`European Journal of Pharmacology 351 1998 53–59
`
`Caliber dependent calcitonin gene-related peptide-induced relaxation in
`rat coronary arteries: effect of Kq on the tachyphylaxis
`Majid Sheykhzade ), Niels C. Berg Nyborg
`Department of Pharmacology, The Royal Danish School of Pharmacy, Uni˝ersitetsparken 2, DK-2100 Copenhagen Ø, Denmark
`
`Received 8 January 1998; accepted 7 April 1998
`
`Abstract
`
`.
`(cid:14)
`The influence of vessel caliber on rat calcitonin gene-related peptide rat-aCGRP -induced responses and the reproducibility of
`rat-aCGRP concentration–response curves were investigated in the left intramural coronary artery of Sprague–Dawley rats. Rat-aCGRP
`–10 M induced concentration-dependent relaxations with a pD -value equal to 8.43 "0.05 ns44 and maximal relaxation
`y7
`(cid:14) y11
`.
`(cid:14)
`.
`10
`equal to 52 "3% ns44 . Both the maximal relaxation and the sensitivity to rat-aCGRP were significantly and inversely correlated
`(cid:14)
`.
`with vessel lumen diameter. The coronary arteries developed tachyphylaxis in response to rat-aCGRP, which was concentration
`dependently decreased by activating the vessels twice with a buffer containing 36 or 125 mM Kq. The rat-aCGRP-curve became fully
`reproducible after activation of the arteries twice with 125 mM Kq. These results indicate a caliber-related dependency of both the effect
`of and sensitivity to rat-aCGRP in intramural rat coronary arteries because the arteries become more sensitive and reactive to rat-aCGRP
`with decreasing caliber. Tachyphylaxis can be avoided by repeated activation with 125 mM Kq. q 1998 Elsevier Science B.V. All rights
`reserved.
`
`2
`
`q
`.
`(cid:14)
`Keywords: CGRP, Calcitonin gene-related peptide ; Vessel caliber; Tachyphylaxis; K ; Coronary artery
`
`1. Introduction
`
`.
`(cid:14)
`Calcitonin gene-related peptide CGRP is a 37-amino
`acid residue peptide generated by alternative splicing of
`(cid:14)
`calcitonin gene transcripts Amara et al., 1982; Rosenfeld
`.
`et al., 1983 . CGRP immunoreactivity is widely distributed
`tissue and co-localized with substance P in
`in neural
`(cid:14)
`.
`sensory nerves in the body Franco-Cereceda et al., 1987b .
`CGRP-containing nerve fibers are found in high density in
`cardiac tissue and they are particularly abundant in the
`atrial muscle and the ventricular vasculature, indicating an
`important function of CGRP in the regulatory processes of
`(cid:14)
`coronary blood flow and myocardial contractility Franco-
`.
`Cereceda et al., 1987b .
`In vitro studies have shown that CGRP causes a posi-
`tive inotropic action in guinea pig atria and potent vaso-
`dilatation in various mammalian coronary arteries
`(cid:14)Franco-Cereceda and Lundberg, 1985; McEwan et al.,
`1986; Franco-Cereceda et al., 1987a; Shoji et al., 1987;
`.
`Prieto et al., 1991a . CGRP can be released during hypoxia
`(cid:14)
`and by low pH levels in the myocardium Franco-Cereceda
`
`)
`
`Corresponding author. Tel.: q45-3537-6777, ext. 546; fax: q45-
`3537-4457; e-mail: mash@mail.dfh.dk
`
`0014-2999r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.
`(cid:14)
`.
`PII S 0 0 1 4 - 2 9 9 9 9 8 0 0 2 9 0 - 8
`
`.
`and Lundberg, 1992 , thus suggesting a vasodilator role
`during ischemic conditions. Recent functional studies in
`rat, porcine and human coronary vascular preparations
`have shown variations in CGRP vasodilator activity and
`tachyphylaxis, depending on the size and the location of
`(cid:14)
`the vessels used Foulkes et al., 1991; Gray and Marshall,
`.
`1991; Ludman et al., 1991; Prieto et al., 1991a . The
`distribution of CGRP-binding sites and receptor subtypes
`has also been shown to exhibit species dependent variabil-
`ity as well as regional variation in different vascular beds
`(cid:14)
`.
`Ludman et al., 1991; Knock et al., 1992 .
`We have previously shown development of tachyphy-
`laxis as well as deviation in CGRP-mediated relaxation
`between epicardial and intramural rat coronary arteries
`(cid:14)
`.
`Prieto et al., 1991a , but a possible variability in the effect
`of CGRP within the intramural coronary arteries was not
`investigated. Since the intramural coronary artery has a
`(cid:14)
`general heterogeneous receptor distribution Nyborg and
`.
`Mikkelsen, 1988; Nyborg, 1991 , we aimed in the present
`study to further investigate the influence of vessel caliber
`on rat-aCGRP-induced responses and the reproducibility
`of rat-aCGRP concentration–response curves in intramu-
`ral ring segments of the left anterior descending coronary
`artery from 3-month-old Sprague–Dawley rats. The in-
`
`EX2219
`Eli Lilly & Co. v. Teva Pharms. Int'l GMBH
`IPR2018-01427
`
`1
`
`

`

`54
`
`M. Sheykhzade, N.C. Berg NyborgrEuropean Journal of Pharmacology 351 1998 53–59
`)
`(
`
`volvement of endothelium in rat-aCGRP-induced relax-
`ation was not investigated in these coronary arteries since
`(cid:14)
`.
`we Prieto et al., 1991a previously have shown that
`rat-aCGRP induces an endothelium-independent relax-
`ation in rat intramural coronary arteries.
`
`2. Materials and methods
`
`2.1. Dissection and mounting
`
`.
`(cid:14)
`Intramural segments 1–2 mm long of the left anterior
`descending coronary artery were isolated from the hearts
`of 3-month-old male Sprague–Dawley rats, as previously
`(cid:14)
`.
`described Nyborg and Mikkelsen, 1985, 1988 . The arter-
`ies were mounted as rings on two 40-mm stainless steel
`wires connected to a force transducer and a micrometer,
`respectively,
`in the organ bath of a double myograph
`(cid:14)
`.
`Mulvany and Halpern, 1977; Mulvany and Nyborg, 1980 .
`This allowed direct determination of the isometric wall
`tension while the internal circumference of the vessels was
`controlled.
`
`2.2. Experimental procedure
`
`centration–response curves were made by precontracting
`the coronary arteries with 10y5 M prostaglandin F
`and
`2 a
`then adding rat-aCGRP to the organ bath in a cumulative
`y7
`(cid:14) y11
`.
`manner 10
`–10 M .
`In protocol A, time-dependent changes in vessel respon-
`siveness were investigated by dividing the organ bath in
`two, which enabled parallel experiments to be performed
`with the two mounted vessels on the myograph. One vessel
`was used to record two consecutive rat-aCGRP concentra-
`y7
`(cid:14) y11
`.
`tion–response curves 10
`–10 M with a 15-min
`wash-out period with PSS between the two curves. The
`other vessel served as time-control and was kept in normal
`PSS. It was used only once to make a rat-aCGRP concen-
`tration–response curve at the same time as the second
`concentration–response curve was made with the first
`vessel.
`In protocol B, the vessels were activated twice for 3
`min with either 36 mM Kq or 125 mM Kq within the
`15-min washout period before the second rat-aCGRP
`concentration–response curve was recorded. The activation
`of vessels with Kq was performed in order to investigate if
`there is a relationship between the concentration of Kq in
`the activating solution and the reduction of rat-aCGRP-
`(cid:14)
`.
`tachyphylaxis. In the third protocol protocol C we used
`the same procedure as protocol B except that the vessels
`were activated twice with 10y5 M 5-HT in order to
`investigate the effect of receptor-mediated contractions on
`the development of tachyphylaxis to rat-aCGRP. The
`contractions induced by prostaglandin F
`were stable
`2 a
`throughout the time needed to make the rat-aCGRP con-
`centration–response curves.
`
`2.3. Drugs
`
`(cid:14)
`Drugs used were rat-aCGRP, 5-hydroxytryptamine 5-
`.
`(cid:14)
`.
`HT HCl Sigma, St. Louis, MO, USA and prostaglandin
`(cid:14)
`.
`w
`, UpJohn, Belgium . 5-HT and rat-aCGRP
`Dinoprost
`F
`2 a
`were dissolved in distilled water. Stock solutions of 5-HT
`10 M and rat-aCGRP 10 M were stored at y208C
`(cid:14) y2
`(cid:14) y4
`.
`.
`and dilutions were made just before experimentation.
`
`2.4. Data analysis and statistics
`
`Vessel responses are expressed as either active vessel
`y1.
`(cid:14)
`wall tension N m , calculated as the increase in vessel
`wall force divided by twice the length of the vessel
`segment, or as a percentage of the response induced by
`prostaglandin F .2 a
`Sensitivity to rat-aCGRP is expressed in terms of
`w
`x.
`pD -values, where pD sylog EC M and is the con-
`(cid:14)
`2
`2
`50
`centration of agonist required to produce half-maximum
`w
`x
`relaxation. EC M was estimated by using iterative non-
`50
`(cid:14)
`linear regression analysis GraphPAD program GraphPAD
`.
`corp, San Diego, CA, USA fitting the data to a sigmoid
`w
`xn
`w
`xn.
`w
`xn
`equation: RrR sA M r A M qEC M , where
`(cid:14)
`max
`50
`is the maximum response developed to the agonist,
`R
`
`max
`
`After mounting, the arteries were equilibrated at 378C
`(cid:14)
`.
`for 30 min in oxygenated 95% O and 5% CO physio-
`2
`2
`(cid:14)
`.
`logical salt solution PSS with the following composition
`(cid:14)
`.
`in mM : NaCl 119, NaHCO 25, KCl 4.7, KH PO 1.18,
`3
`2
`4
`MgSO P7H O 1.17, CaCl P2H O 21.5, ethylene diamine
`4
`2
`2
`2
`(cid:14)
`.
`tetraacetic acid EDTA 0.027 and glucose 5.5; pH was
`adjusted to 7.4. The vessels were then stretched to their
`optimal lumen diameter l s0.9 =l
`, where l
`is an
`1
`100
`100
`estimate of the diameter the vessel would have under a
`y2 . (cid:14)
`(cid:14)
`passive transmural pressure of 13.3 kPa N m
`100 mm
`.Hg in order to obtain optimal conditions for active tension
`(cid:14)
`.
`development Nyborg et al., 1987 . Each experiment was
`initiated by contracting the vessels repeatedly with K-PSS
`(cid:14)similar to PSS except that NaCl was replaced by KCl on
`.
`an equimolar basis until reproducible wall tensions were
`recorded. The maximal contractile response of the vessels
`(cid:14)
`.
`DT
`was then determined by measuring the differences
`max
`(cid:14)
`in vessel wall tension newton per meter of vessel wall, N
`y1.m
`when the vessel was maximally contracted with
`y5
`y5
`(cid:14)
`.
`K-PSS to which 10 M serotonin 5-HT and 10 M
`prostaglandin F were added and when maximally relaxed
`2 a
`2q
`(cid:14)
`.
`in Ca
`- free PSS Nyborg, 1991; Prieto et al., 1991a .
`Ca2q-free PSS was similar to PSS except that CaCl was
`2
`(cid:14)
`replaced with 0.01 mM ethylene glycol-bis b-aminoethyl
`X
`X
`.
`(cid:14)
`.
`ether -N, N, N , N -tetraacetic acid EGTA . Vessels were
`(cid:14)
`accepted only if the maximal active pressure calculated
`according to Laplace’s law: D P s2 = DT rl
`.
`ex-
`ceeded 9 kPa.
`Three protocols were used to investigate the develop-
`ment of tachyphylaxis and the rat-aCGRP response in
`relation to caliber in 10y5 M prostaglandin F -precon-
`2 a
`tracted coronary arteries. In all protocols, rat-aCGRP con-
`
`max
`
`1
`
`max
`
`2
`
`

`

`M. Sheykhzade, N.C. Berg NyborgrEuropean Journal of Pharmacology 351 1998 53–59
`)
`(
`w xA M is the concentration of agonist and n is a curve-fit-
`
`(cid:14)
`.
`ting parameter, the Hill coefficient Kenakin, 1986 . Re-
`sults are given as means"S.E.M., nsnumber of ves-
`(cid:14)
`.
`sels . Differences between mean values were analyzed by
`using a two-tailed Student’s t-test for paired or unpaired
`observations where appropriate. One-way analysis of vari-
`(cid:14)
`.
`(cid:14)
`ance ANOVA was used to compare the contractions %
`.
`of the first and second rat-aCGRP-curves. The
`of DT
`max
`level of significance was for all tests set to P-values less
`than 0.05.
`
`1
`
`3. Results
`
`3.1. Effect of time on ˝essel response to rat-aCGRP
`
`In protocol A, in which the time dependency of the
`rat-aCGRP-response was investigated, there was no sig-
`nificant difference either in maximal rat-aCGRP-induced
`relaxation or in sensitivity to rat-aCGRP between the first
`(cid:14)
`.
`curves Fig. 1B . The maximal relaxation obtained for the
`first curves was 58 "4% ns10 vs. 57 "6% ns10
`(cid:14)
`.
`(cid:14)
`.
`and the corresponding sensitivity was 8.50 "0.10 ns10
`(cid:14)
`.
`vs. 8.35 "0.08 ns10 for the twice-exposed vessels and
`(cid:14)
`.
`the time-control vessels, respectively. However, the maxi-
`mal rat-aCGRP-induced relaxation in the twice-exposed
`(cid:14)
`.
`(cid:14)
`vessels was significantly P -0.05 reduced 44 "4% n
`s10 in the second rat-aCGRP-curve Fig. 1A . Mean
`.
`(cid:14)
`.
`(cid:14)
`.
`of the coronary arteries was 208"10
`lumen diameter
`l
`mm ns10 for twice-exposed vessels and 209"9 mm
`(cid:14)
`.
`ns10 for the time-control vessels, respectively.
`(cid:14)
`.
`
`1
`
`3.2. Effect of K q- and 5-HT-induced contraction on the
`reproducibility of rat-aCGRP-induced responses
`
`When the coronary arteries were washed twice with
`PSS containing 5.9 mM Kq, they developed a pronounced
`
`55
`
`1
`
`tachyphylaxis to rat-aCGRP, as indicated in Fig. 2A. The
`(cid:14)
`maximal response to rat-aCGRP was significantly P -
`0.01 reduced 44 "4% ns16 in the second curve, the
`.
`(cid:14)
`.
`maximal relaxation being 48 "5% ns16 and 27 "4%
`(cid:14)
`.
`ns16 for the first and second curves, respectively. The
`(cid:14)
`.
`sensitivity of the vessel to rat-aCGRP was not altered, the
`ns16 and 8.20 "0.07
`(cid:14)
`.
`pD -value being 8.32 "0.10
`2
`ns14 for the first and second curves, respectively.
`(cid:14)
`.
`(cid:14)
`.
`Mean lumen diameter
`of the coronary arteries was
`l
`247"18 mm ns16 .
`(cid:14)
`.
`When the vessels were activated twice with 36 mM Kq
`between the two rat-aCGRP dose–response curves, there
`was still a significant P -0.05 22 "7% ns6 reduc-
`(cid:14)
`.
`(cid:14)
`.
`(cid:14)
`tion in the maximal rat-aCGRP-induced relaxation Fig.
`2B . Maximal relaxation was 54 "11% ns6 and 40 "
`.
`(cid:14)
`.
`8% ns6 for the first and second curves, respectively.
`(cid:14)
`.
`Again, the vessel sensitivity to rat-aCGRP was not signifi-
`cantly altered, the pD value being 8.50 "0.10 ns6
`(cid:14)
`.
`2
`and 8.32 "0.07 ns6 for the first and second curves,
`(cid:14)
`.
`(cid:14)
`.
`respectively. Mean lumen diameter
`of the coronary
`l
`arteries was 236"20 mm ns6 .
`(cid:14)
`.
`When 125 mM Kq was used in the activating solution,
`there was no significant reduction in the maximal rat-
`aCGRP-induced relaxation between the first and the sec-
`ond curves. Maximal relaxation was 53 "9% ns6 for
`(cid:14)
`.
`the first curve and 50 "9% ns6 for the second curve
`(cid:14)
`.
`(cid:14)
`.
`Fig. 2C . There was no significant difference in sensitivity
`to rat-aCGRP between the first and the second curves,
`pD value being 8.47 "0.15 ns6 and 8.32 "0.15 ns
`(cid:14)
`.
`(cid:14)
`2
`.6 for the first and second curve, respectively. Mean lumen
`of coronary arteries was 249"25 mm ns
`(cid:14)
`.
`(cid:14)
`diameter
`l
`1
`.6 .
`When the data from these three experiments were com-
`bined, an inverse linear relationship rs0.74; Ps0.0001,
`(cid:14)
`ns28 was found between the concentration of K in the
`q
`.
`activating solution and the reduction in maximal rat-
`(cid:14)
`.
`aCGRP-induced relaxation Fig. 3 .
`
`y5
`y7
`(cid:14) y1 1
`.
`Fig. 1. The effect of time on rat-aCGRP concentration–response curve 10
`–10 M recorded in 10 M prostaglandin F -precontracted coronary
`2 a
`arteries from Sprague–Dawley rats using protocol A. Closed symbols are used for twice-exposed vessels 1st Curve, v; 2nd Curve, % and open circle is
`(cid:14)
`.
`used for time-control vessels. Points represent mean values for 10 vessels and vertical bars indicate "S.E.M. where this value exceeds the size of symbol.
`(cid:14) y5
`.
`10 M just before the vessels were challenged with rat-aCGRP.
`Responses are given as percentages of the initial response to prostaglandin F
`2 a
`
`3
`
`

`

`56
`
`M. Sheykhzade, N.C. Berg NyborgrEuropean Journal of Pharmacology 351 1998 53–59
`)
`(
`
`y7
`(cid:14) y1 1
`q
`.
`Fig. 2. The effect of different concentrations of extracellular K on the reproducibility of rat-aCGRP concentration–response curves 10
`–10 M
`recorded in 10y5 M prostaglandin F -precontracted coronary arteries from Sprague–Dawley rats using protocol B. Closed circles represent the first
`2 a
`ns16 , B K-PSS containing 36 mM K
`ns6 and C
`q (cid:14)
`q (cid:14)
`(cid:14)
`.
`. (cid:14) .
`.
`(cid:14) .
`curves and closed triangles represent the second curves. A PSS containing 5.9 mM K
`ns6 . Points represent mean values and vertical bars indicate "S.E.M. where this value exceeds the size of symbol.
`q (cid:14)
`.
`K-PSS containing 125 mM K
`(cid:14) y5
`.
`10 M just before the vessels were challenged with rat-aCGRP.
`Responses are given as percentages of the initial response to prostaglandin F
`2 a
`
`In protocol C, in which 10y5 M 5-HT was used as
`intermediary activator, the maximal rat-aCGRP-induced
`(cid:14)
`.
`relaxation was significantly P -0.01 reduced 57 "4%
`ns6 , from 55 "6% ns6 in the first curve to 24 "4%
`.
`(cid:14)
`.
`(cid:14)
`ns6 in the second curve Fig. 4 . The vessel sensitivity
`(cid:14)
`.
`(cid:14)
`.
`to rat-aCGRP was not significantly altered, the pD -value
`2
`being 8.75 "0.12 ns6 and 8.50 "0.10 ns6 for the
`(cid:14)
`.
`(cid:14)
`.
`
`1
`
`first and second curves, respectively. Mean lumen diame-
`of coronary arteries was 202"15 mm ns6 .
`(cid:14)
`.
`(cid:14)
`.
`ter
`l
`The contractions induced by 36 mM Kq, 125 mM Kq
`and 10y5 M 5-HT as percentage of DT
`were 36 "4%
`max
`ns6 , 88 "3% ns6 and 71 "6% ns6 , respec-
`(cid:14)
`(cid:14)
`.
`(cid:14)
`.
`.
`q
`q
`(cid:14)
`tively P -0.001; 36 mM K vs. 125 mM K , P -0.05;
`125 mM Kq vs. 5-HT-induced contraction, one-way
`.
`ANOVA with Bonferorroni correction .
`
`3.3. Relation between ˝essel caliber and maximal rat-
`aCGRP-induced relaxation
`
`The compiled average maximal relaxation induced by
`rat-aCGRP in prostaglandin F -precontracted arteries was
`2 a
`52 "3% ns44 . The sensitivity pD -value of the ves-
`(cid:14)
`.
`(cid:14)
`.
`
`2
`
`q (cid:14)
`.
`Fig. 3. Relationship between the concentration of K
`closed circles in
`the activating solution and the reduction in maximal rat-aCGRP-induced
`relaxation between the first and second curves recorded in 10y5 M
`prostaglandin F -precontracted coronary arteries from male Sprague–
`2 a
`Dawley rats. Open circle shows reduction in maximal rat-aCGRP-relaxa-
`tion in the vessels contracted with 10y5 M 5-HT in the interval between
`the recording of two rat-aCGRP-curves. Points represent mean values of
`6–16 vessels and vertical bars indicate "S.E.M. where this value
`exceeds the size of symbol. Reduction in maximal rat-aCGRP-induced
`relaxation is given as percentage change in maximal relaxation from the
`first curve to the second curve.
`
`Fig. 4. The effect of contraction elicited by 10y5 M 5-HT on the
`(cid:14) y1 1
`reproducibility of rat-aCGRP concentration–response curves 10
`–
`y7
`.
`10 M recorded in prostaglandin F -precontracted coronary arteries
`2 a
`from Sprague–Dawley rats using protocol B. Closed circles represent the
`first curves and closed triangles represent
`the second curves. Points
`represent mean values for 6 vessels and vertical bars indicate"S.E.M.
`where this value exceeds the size of symbol. Responses are given as
`percentages of the initial response to 10y5 M prostaglandin F
`just
`before the vessels were challenged with rat-aCGRP.
`
`2 a
`
`4
`
`

`

`M. Sheykhzade, N.C. Berg NyborgrEuropean Journal of Pharmacology 351 1998 53–59
`)
`(
`
`57
`
`y5
`(cid:14) .
`.
`(cid:14)
`Fig. 5. Relationship between the lumen diameter and maximal rat-aCGRP-induced relaxation A and sensitivity to rat-aCGRP B in 10 M
`prostaglandin F -precontracted coronary arteries ns44 from male Sprague–Dawley rats.
`(cid:14)
`.
`
`2 a
`
`sels to rat-aCGRP was 8.43 "0.05 ns44 and the mean
`.
`(cid:14)
`of arteries was 231"9 mm ns44 .
`(cid:14)
`.
`(cid:14)
`.
`lumen diameter
`l
`1
`The mean vessel response to prostaglandin F was 1.56 "
`2 a
`ns44 , equal to 75 "1% ns44 of the
`y1 (cid:14)
`.
`(cid:14)
`.
`0.08 N m
`(cid:14)
`.
`maximal contractile response of vessels DT
`, which
`max
`ns44 .
`y1 (cid:14)
`.
`was 2.08 "0.12 N m
`Further analysis of these data showed that there was a
`significant correlation between the arterial lumen diameter
`(cid:14)
`.
`relative to the maximal rat-aCGRP-induced relaxation
`and the vessel sensitivity to rat-aCGRP, which was best
`fitted to an exponential decay function rs0.87; P -
`(cid:14)
`line function rs0.70;
`0.0001; ns44 and a straight
`.
`(cid:14)
`P -0.0001; ns44 , respectively Fig. 5A and B .
`.
`(cid:14)
`.
`The caliber of the coronary arteries had no influence
`(cid:14)
`.
`neither on the level of preconstriction tone % of DTmax
`rs0.02; Ps0.89; ns44
`.
`(cid:14)
`induced by prostaglandin F
`2 a
`nor on the attenuation of the maximal rat-aCGRP-induced
`response in vessels kept in PSS in the interval between
`rs
`(cid:14)
`recording of the two concentration–response curves
`0.01; Ps0.97; ns16 , since both regression lines had
`.
`slopes not significantly different from zero.
`
`4. Discussion
`
`The present study shows that relaxations induced by
`rat-aCGRP in rat intramural coronary arteries as well as
`vessel sensitivity to rat-aCGRP depends on the caliber of
`the vessels, as both response and sensitivity increased with
`deceasing vessel diameter. Our finding indicates that CGRP
`exerts its major vasodilator effects in coronary arteries
`with lumen diameters less than 200 mm, in which the
`(cid:14)
`major part of the precapillary flow resistance resides Chil-
`.
`lian, 1988 . Vessel caliber has been shown to influence the
`response of rat coronary arteries to other vasoactive com-
`pounds. The contractile response to 5-HT decreases with
`(cid:14)
`.
`decreasing vessel caliber Nyborg and Mikkelsen, 1988
`and is possibly related to the 5-HT receptor reserve in
`
`.
`(cid:14)
`these arteries Nyborg, 1991 . Noradrenaline causes con-
`traction in rat epicardial and dilation in intramural small
`(cid:14)
`.
`arteries Nyborg, 1990 , and the magnitude of dilatation
`induced by the Kq channel opener, levcromakalim, in-
`(cid:14)
`.
`creases with decreasing vessel diameter Sato et al., 1994 .
`A recent study has demonstrated that the mechanisms of
`the coronary microvascular responses to pertussis toxin-
`sensitive G protein activation depends on vessel caliber
`(cid:14)
`.
`Komaru et al., 1997 . These studies indicate that receptor
`distribution, ion channels and second messenger systems
`may be finely adjusted within the coronary circulation,
`probably reflecting the physiological demands on the vas-
`cular segment.
`Tachyphylaxis or attenuation of the vascular response to
`peptides is often encountered. We have previously de-
`scribed this for neuropeptide Y-induced contractions and
`(cid:14)
`rat-aCGRP-induced relaxations in Wistar rats Prieto et
`.
`al., 1991a,b . Tachyphylaxis to CGRP also develops in
`(cid:14)
`porcine coronary arteries Gray and Marshall, 1991; Mar-
`.
`shall, 1992 . Our data showed that the development of
`to rat-aCGRP was pronounced during
`tachyphylaxis
`recording of the second concentration–response curve for
`rat-aCGRP in the experiments where the vessels were
`kept in buffer containing a normal concentration of 5.9
`mM Kq. The development of tachyphylaxis was not time-
`dependent or dependent on the caliber of the vessels.
`However, activation of the arteries twice with 36 or 125
`mM Kq between recording of the two rat-aCGRP concen-
`tration–response curves reduced the development of tachy-
`phylaxis and the rat-aCGRP concentration–response curve
`became fully reproducible after activation of the arteries
`twice with 125 mM Kq. These findings could indicate that
`rat-aCGRP mediates its vasodilator action partly via a
`mechanism that is inactivated during the first exposure of
`the vessels to rat-aCGRP and which is reactivated by the
`depolarization of the smooth muscle cell membrane with
`Kq. This is further supported by our finding that 5-HT was
`without an opposing effect on the development of tachy-
`
`5
`
`

`

`58
`
`M. Sheykhzade, N.C. Berg NyborgrEuropean Journal of Pharmacology 351 1998 53–59
`)
`(
`
`phylaxis even though the magnitude of contractions in-
`duced by this receptor-mediated agonist was close to that
`induced by 125 mM Kq, thus indicating that it is not the
`contraction itself that opposes the development of tachy-
`phylaxis.
`The tachyphylaxis to CGRP and capsaicin was pre-
`vented in guinea pig hearts by using colchicine, an in-
`hibitor of tubulin polymerization and internalization of
`(cid:14)
`.
`membrane-bound proteins Franco-Cereceda, 1990 .
`It
`could be speculated that membrane depolarization andror
`high extracellular Kq concentrations mimic the action of
`colchicine, preventing internalization of the CGRP recep-
`tors on the vascular smooth muscle cells in the rat coro-
`nary arteries.
`It is known that CGRP mediates its vasorelaxing effect
`through different and combined mechanisms of action
`(cid:14)
`.
`Kline and Pang, 1997 . The direct action of CGRP on
`vascular smooth muscle is generally thought to be medi-
`ated by an increase in the intracellular cAMP level, which
`subsequently alters protein kinase A-dependent kinase ac-
`tivity and hence causes relaxation of smooth muscle cell
`(cid:14)Crossman et al., 1987; Edwards et al., 1991; Gray and
`.
`Marshall, 1991 . Repeated application of CGRP can there-
`fore bring about desensitization of one or two biochemical
`pathways involved in vasodilator mechanisms.
`Patch-clamp and electrophysiological experiments have
`shown that CGRP can hyperpolarize rabbit arterial smooth
`muscle cell membrane by opening Kq channels which
`ATP
`(cid:14)
`are inhibited by glibenclamide Standen et al., 1989; Nel-
`.
`son et al., 1990; Kitazono et al., 1993 . Since the graded
`depolarization of the smooth muscle cell membrane with
`36 or 125 mM Kq reduced the attenuation of the rat-
`aCGRP-induced response in the coronary arteries,
`this
`procedure could reactivate ion channels in the smooth
`muscle cell membrane. We have no indication of which
`type of channels these are, but they are not likely to be
`Kq channels or other Kq channels since glibenclamide
`ATP
`and high concentrations of tetraethylammonium have no
`inhibitory effect at all on CGRP-induced relaxations in
`(cid:14)
`intramural coronary arteries in Wistar rats Prieto et al.,
`.
`1991a .
`The sensitivity of Sprague–Dawley coronary arteries to
`rat-aCGRP in our experiments was similar to that reported
`in a previous study with coronary arteries from Wistar rats
`(cid:14)
`.
`Prieto et al., 1991a and close to that of many other
`(cid:14)
`vascular preparations Foulkes et al., 1991; Lei et al.,
`.
`1994 . The inverse linear correlation between response and
`sensitivity to rat-aCGRP and vessel caliber may indicate
`that the rat-aCGRP-induced response is limited by the
`receptor density andror intermediary intracellular messen-
`gers in the arteries used in our study.
`In conclusion, rat coronary arteries become more sensi-
`tive and reactive to rat-aCGRP with decreasing caliber,
`indicating that CGRP exerts its major vasodilator action in
`the flow-regulating coronary arteries. The results also show
`that only repeated exposure of the arteries to 125 mM Kq
`
`can overcome the development of tachyphylaxis; however,
`the mechanism behind the development of tachyphylaxis
`and the caliber-dependent response to rat-aCGRP remains
`to be elucidated in intramural coronary arteries.
`
`Acknowledgements
`
`This work is supported by the Danish Heart Foundation,
`Grant No. 96-1-3-64r22386 and Novo Nordisk Research
`Foundation.
`
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