Pharmacological coupling and functional role for CGRP
`receptors in the vasodilation of rat pial arterioles
`
`KI WHAN HONG, SUNG-EUN YOO, SUNG SUK YU,
`,JUNG YOON LEE, AND lWUNG YONG l{Hll\11
`Department of Pharmacology, College of Medicine, Pusan National University,
`Pusan 602-739; Center for Biofunctional Molecules, Pohang; and
`Korea Research Institute o/ Chemical Technology, Dajeon, Korea
`
`Hong, Ki Whan, Sung-Eun Yoo, Sung Suk Yu, Jung
`Yoon Lee, and Byung Yong Rhim. Pharmacological cou(cid:173)
`pling and functional role for CGRP receptors in the vasodila(cid:173)
`tion of rat pial artoriolos. Am . .I. Ph._ysiol. 270 (Heart Circ.
`Physiol. 39): H317-H323, 1996.- Tn this study, we investi(cid:173)
`gated tho sig11al transduction underlying the vasodilator
`action of calcitonin gene-related peptide (CGRP) in the rat
`pial arterioles. In an in vivo experiment, changes in pial
`arterial diameters (20.2 ::':: L!:l µm ) were observed under
`suffusion with mock cerebrospinal fluid containing CGRP
`(l0 - 9- 10- 7 M) directly through a closed cranial window.
`Changes in intracellular adenosine 3' ,5' -cyclic · monophos(cid:173)
`phaie (cAlvIP) accumulation in reHponse to CGRP and levcro(cid:173)
`makalim were measured in the pial arterioles in an in vitro
`experiment. CGRP-induced va:-;ndilatinn and cAMP prnduc•
`tion were significantly inhibited by specific CGHP antibody
`serum and CGRP-(8- 37) fragment, suggesting involvement.
`of the CGRP1 receplor sublype. Vasodilation and increase in
`cAMP production evoked by CGRP were inhibited not only by
`glibenclamide (ATP-sern;itive K+ channel hlocker) but, also by
`charybdotoxin (large-conductan~e Ca2+-activated K- channel
`blocker), but this was not the case for the isoproterenol(cid:173)
`inrlnc:ed vasodilat:ion and cAMP production. 'l'hc~s,~ findings
`implicate the A'I'P-RenRitive K
`channels and the large(cid:173)
`conductance Ca2 1 -activated K I channels in the CGRP recep(cid:173)
`tor-coupled cAivfP product.ion for vasodilation. Further study
`is required to identify whether the cAMP-dependent K
`channel activation is related to CGRP-induced vasorelax(cid:173)
`ation of the rat pial arterioles.
`calciwnin gen e -relate d peptide; adenosine 3' ,5 '-cyclic mono(cid:173)
`phosphnte; potassium channels
`
`pial arterioles, and the vasodilatory effect of CGRP is
`associated with an activation of ATP-sensit ive K + chan(cid:173)
`nels (14, 18). Recently, Kitazono et al. (16) reported that
`activation of ATP-sensitive K + channels in the rat
`basilar artery was mediated by an adenosine 3 1 ,5' -
`r.yr.lir monophosph:=itP. (r.AMP)-clP.pP.nclP.nt mer.h:=inism
`in the CGRP-imluced vasuuilatiun. On Lhe uLher hauu,
`the vasodilator response to CGRP has been corrclut.cd
`with increases in cAMP and guanosine 3 ',5'-cyclic
`monophosphate (7, 17). However, the clependency of
`CGRP-induced vasodilation on the endothelium is re(cid:173)
`portedly not consistent among species and among or(cid:173)
`gans (2, 7, 12).
`In the present study, we investigate th e important
`target of signal transduction mechanisms underlying
`the vasodilator action of CG.RP in the rat pial arteri(cid:173)
`oles. We have measured two pararm:iers in ihe re(cid:173)
`sponse to CGRP: 1) the changes in the luminal diam(cid:173)
`P.tP.r of pi:=il :=irtP.riolP.s in thP. in vivo PxpPrimP.nt and 2)
`the changes in cAMP production in the pial arterioles in
`the in vitro experiment in the absence and presence of
`K+ channel blockers, because it has been document ed
`that the relaxant effects of CGRP on the vascular
`smooth muscles are inhibited by glibenclamide (25).
`The results induced by CGRP were compared with
`those evoked by isoprotcrenol and levcromakalim. ln
`particular, the biochemical action (activation of adenyl
`cyclase) of CGRP was corn~lated with ih1 functional
`effect (vasodilation) in the rat pial arterioles.
`
`A GROWING BODY Of EVIDENCE has demonstrated that
`culcitonin gone rolutod peptide (CGRP) exerts an ox
`tremely potent vasodilating effect on the cerebral ar(cid:173)
`tery in vitro (7, 29) and on the pial artery in Ritu (20,
`34). In tho cerebral vessols, CGRP-immunoroaciivo
`nerve fibers are demonstrated in all major arteries and
`cortical arterioles, including human pia1 arterioles (8).
`CGRP has been proposed a s a candidate neurotransmit(cid:173)
`ter involved in nonsympathetic neurogenic vasodila(cid:173)
`tion oftlrn rPrPhr:=i 1 v:rnr.11 l:=ir hPrl (1, ~!-))_
`It is widely known that activation of ATP-sensitive
`K+ channels is an important mechanism of many
`va!mdilators (24, 26). The vasodilator effect of CGRP is
`mediated in part, via activation of ATP-sern;itive K'
`channels (25) in contrast to other arteries (27). Re(cid:173)
`cently, we reported that CGRP is implicated in cerebral
`autoregulation; that is, CGRP, which is reflexly re(cid:173)
`leased from perivascular sensory fibers in response to a
`t ransie nt hypote n sion, m ediates the vasodila tion of rat
`
`l\'IATERIAL!-l AND METHODS
`
`Preparation o(Amma1s
`
`Spr<!gue-Dawley r·ats (2G0-300 g) were anestheti,;ed with
`urethan (1 g/kg ip) and placed on a heating pad to maintain a
`curn;Laul botly LemµeraLure. Afler a Lrad1eoi:iLomy wa:; per(cid:173)
`formed, each rat was ventilated with a respirator (model 683,
`Harvard) with room air. The left femoral artery was cannu(cid:173)
`lated with PE-50 polyethylene tubing for m onitoring blood
`pressure (Statham P23D pressure transducer). Arterial blood
`was collected through the left carotid artery before and after
`installation ofa cranial window for blood gas and pH determi(cid:173)
`nation (STAT Profile 3, Nova Biomcdicals). The mean arterial
`blood gases and pH determined during ex periments were as
`follows: pH, 7.37 .:!:: 0.01; arterial Pco2, 31.3 .::. 1.6 mmHg;
`artc \riiil Po2, ~8.0 ::':: 2.2 mmHg. Rc\cb1l t.l\mpc\ratun\ was
`monitored continuously and kept constant {37 .c.. O.G°C) with a
`heating pad.
`
`Pial microvessels were visualized through an implanted
`cranial window, as described previously (14). Briefly, the h ead
`
`0363-6 135/96 S5. 00 Copyright © 1996 the Ame rican Phy8iological Society
`
`H3l7
`
`

`

`H318
`
`CGRP AND RAT PlAL ARTERIOLE
`
`was fixed in the prone position with a stereotaxic apparatus
`(Stoelting), and a square (5 X 5-mm) craniotomy was made
`over Lhe righL parietal corlex. The durn ww; opened, and Lhe
`buIT hole ,vas covered with warmed mineral oil during the
`operation. Pial precapillary micruvessels (15-25 µm) were
`visualized through the implanted cranial window. Cerebral
`micruvessels were allowed lo equilibrate for 60 min after
`installation of the cranial window. The window field was
`suffused with mock cerebrospinal fluid (CSF) at 0.3 ml/min.
`The image of pi.al vessels was captured wi.th a CCD videocam(cid:173)
`era (Sanyo. VDC 8900) through a stereoscope (model SMZ-2T,
`Nikon) and fed to a television monitor for direct observation,
`llnr'I t.hP. r.ll lihP.r Wl'IS mP.l'ISllrP.cl m;ing ri \Vin th nnn ly:u~r (H ama(cid:173)
`matsu) at X 480 magnification. The composition (mM) of the
`mock CSF was as follows: 125 NaCl, 3.5 KCl, 1.3 CaC12, 1.1
`MgCl2, am! 2f> NaHCO3. The iuLracranial pressure was
`maintained constant at 5-6 mm.Hg throughout the experi(cid:173)
`ment by ac,ljusting- the height of the free end of the plastic
`tubing, which was connected to the outlet of the window. In
`each rat, only one arteriole was observed wider the window.
`
`Protocols of In Vivo Experiment
`
`1) We identified the normal autoregulatory response of the
`pial artery to a decline in arterial blood pressure by bleeding
`the blood into the reservoir and to a rise in blood pressure by
`infusing the blood under sutfosion with mock CSF ovr.r the
`cerebral cortical surface. 2) We observed the vusodilutor
`responses of the resting pial vessels to CGRP suffused with
`the mock CSF. Each concentration of drug was suffused over
`th,1 cort.ir:al s11rfnce for ~ min. 8) ThP. inhihitory P.ffoct.s of
`CGRP antibody scrum (1:1,000) and CGilP-(8- 37) fragment
`(10 8 M) on the CCRP-induced vasodilation were identified.
`The change in luminal diameLer wa~ moniLured with a width
`analyzer every 4 s. 4) Furlhermore, to see the inhibitory
`effects of the K' channel blockers glibenclamide and charyb(cid:173)
`dotoxin, they were applied from 30 min before suffusion of
`CGRP or other agonists.
`
`Cyclic Nucleotide Determination
`
`Rats (Sprague-Dawley, 250- :l00 g) were anesthetized with
`ether and killed by bleeding from carotid arteries. By direct
`observation under a stereoscope, the pial artery and its
`branches (20-30 µm diameter) were dissected from the
`surface of the cerebral cortex, the adhering cxtravascular
`tissue was 1·emoved, and the intraluminal cell components
`were cleaned by perfusion with mock CSF. Adenyl cyclase
`activity was measured on the pial arterioles, as described by
`Edwards ct al. (9). The tissues were subjected to freezing and
`t.h;nving t.wic.P. in liqnid nit.rogP.n ::md fin:=i lly st.orP.n ovPrnight,
`in liquid nitrogen. 'l'he tissues were incubated for 30 min in
`the working buffer consisting of (in mM) 100 tris(hydroxy(cid:173)
`methyl)aminomethane, 4.0 MgC12, 0.2f> ~DTA, 1.0 3-isobutyl-
`1-methylxanthine, 0.3 ATP, 0.1 guanosine 5'-triphosphate,
`and 20 phosphocreatine and 200 U/ml creatine kinase (pH 7.4
`at 30°C) without and with drugs. The incubation was stopped
`by addition of 0.2 N HCl (50 µI) and freezing in liquid
`nitrogen. ThereafLer, the pH of the buffer was readjusted by
`addition of0.::! N NaOH and sodium acetate buffer. cAMP was
`measured with a radioimmunoassay kit purchased from
`AmP.rsham Life Science Products. The activity of adenyl
`cyclase was expressed as femtomolcs of cAMP per square
`millimeter of arteriole surface per 30 min.
`
`Drugs
`
`bovine serum albumin to make a stock solution of 0.1 mM.
`Rabbit antiserum to rat CGRP (Cambridge Research Bio(cid:173)
`chemical:;) was J.iluLeJ. wiLh muck CSF. GliLeuclamide (Sigma
`Chemical) was sonicated in 1 ml ofNaOH (0.1 N) and diluted
`with 5% glucose to make a stoc.:k solution of 10 mM. Isuproter(cid:173)
`enol HCI and forskolin were purchased from Sigma Chemical.
`Levcromakalim was donated by Korea Research Institute of
`Chemical Technology.
`
`Statistics
`Results are expressed as means ..:_ SE. Statistical signifi(cid:173)
`cance was determined by Student's t-test between two groups
`in Lhe measurement uf cAl'vlP levels, arn.l analysis uf variauce
`was used for comparisons of the results ofvasodilators in the
`absence and presence of antagonists. P < 0.05 was considered
`to be statistically significant.
`
`RESULTS
`
`In control rats, mean arterial blood pressure was
`115. 7 ±: 2.5 mmHg (n = 48) und the resting diumctcr of
`pial arteriole was 20.2 + 1.9 pm, which was constant
`throughout the experiment unless there was bleeding
`or an infusion of blood.
`Table 1 compares effects of CG RP and levcromakalim
`on the changes in luminal diameter of pial arterioles.
`When cortical surface was suffused with mock CSF
`containing CGRP (10 9- 10 7 M) and levcromakalim
`(10- 7 and 10- 6 M), hy increasing each concentration,
`the diameter of µial arLerioles was remarkably in(cid:173)
`creased in a concentration-dependent manner (Fig. 1,
`Tabh~ 1). After 30 min of continuous suffusion with
`normal mock CSF, the luminal diameter returned
`almost to the original control value (21.5 ±: 3.1 µm ). The
`maximum responses occurred at 10- 7 M CGRP and
`10 6 M levcromakalim. The maximum diameters were
`28.0 :t 2.2 (n ~ 15, 38.6%) and 27.4 ±: 2.2 µm (n = 4,
`38.4%), r espective ly.
`
`C:hangP..<1, in TJiamP.tP.r in ln Vinn ExpP.riment
`
`Effects of CGRP antibody serum and CGRP-(8- 37).
`The CCRP-induced vasodilation was significantly inhib(cid:173)
`ited under suffusion with mock CSF conLaining CGRP
`antibody serum (1:1,000) and CGRP-(8-:37) fragment
`(10 8 M) from 30 min before Lhe experiment (Fig. 2).
`Application of CGRP antibody serum or CGRP-(8-37)
`
`Table 1. Concentration-dependent increase in diameter
`nf pial arterinleR under suffza;ion with mock CSF
`containing CORP and leucromalmlim in mls
`
`Cuncen~raLion, M
`
`Diameter Control
`
`n
`
`10- 9
`
`10- 0
`
`10-7
`
`10- 0
`
`CGRP
`
`20.2 ±: 1.9 16 19.9 ±: 2.1 23.7::: 2.F 28.0 ±: 2.2,:,
`-1.5
`17 .3
`38.6
`Leucromakalim
`
`19.8 ::t:: 5.5 4
`
`22.6 ::t:: 6.P 27.4 = 2.2''
`11.1
`:38.1
`
`µm
`%.1
`
`µm
`';{:.~
`
`CORP (Sigma Chemical), CGRP-(8-37) (Peninsula), and
`charybdotoxin (Peptide TnstitutP.) wer e dissolved in 0.1 %
`
`Values are mean;; +- SE; n, nu. of experimenL::;. CSF, ce,.ebrospinal
`fluid; CGH.I'. calc.ilonin gc.nc.-n~lalr,d peptide. * /' < O.Oii vs. control.
`
`

`

`CGRP AND RAT PIAL ARTERIOLE
`
`H319
`
`t
`-9
`
`G)
`
`80 A
`... 40
`1i
`E 0
`'°
`~-40
`~
`8,80
`C
`~40
`0
`"<fl 0
`
`CGRP, log M
`
`-40
`Fig. 1. Concentration-dependent increase in diameter of ral pial
`arterioles under suffusion with mock cerebrospinal fluid contriining
`calcitonin gene-related peptide (CCRP, 10-9-10-7 M, n - 15; A) and
`levcromakalim (L-CRK, 10 7 and 10 6 M, n - 4; B ). Values are
`means= SE.
`
`l•CRK,109 M
`
`fragment itself did not signifir.:rntly ::itfP.r.t. t.hP. vP.ssr.l
`diameter (Table 2).
`Effect of K' channel antagonists on CG RP-induced
`vasodilatinn. The vasodilation induced by CGRP ( 10- 9_
`10 ; M) and levcromakalim (10 7 and 10 (i M), a K
`channel opener, was significantly inhibited under previ(cid:173)
`ous suffusion with glibenclamide (lo-r, M), an ATP(cid:173)
`sensitive K - channel antagonist, or charybdotoxin (3 X
`10-7 M), a large-conductance Ca2 -activated K ' chan(cid:173)
`nel antagonist, respectively (Figs. 3 and 1). Suffusion
`with mock CSF containing glibenclamide or charybdo(cid:173)
`toxin from 30 min before Lhe experiment Jiu nut aired
`the resting diameter of pinl arteries (Table 2). These
`results implicate the Ca2+ -activated K+ channels and
`
`Table 2. Effects of xlibenclnmide cuul chwybdotoxin,
`K· channel blockers, and CGRP-(8-37) and CORP
`antibody serum, CGRP antagonists, on diameter
`of' rat pial arterioles
`
`Antagonists
`
`Glibenclamide
`(10 " M)
`Charybdotoxin
`(3 X 10 'M)
`CGRP-(8-37)
`(10 RM)
`CGRP antibody serum
`(1:1,000)
`
`11
`
`8
`
`9
`
`5
`
`4
`
`Control
`Diameter.
`µm
`
`Change in
`Diameter,
`µm
`
`%Change
`in
`Diameter
`
`21.1 :t 4.4
`
`18.7 :t 3.2
`
`-1.3 = 1.4
`
`20.9:!: 3.2
`
`19.5 :!: 2,2
`
`-3.3 = 4.7
`
`17.4:': 1.8
`
`18.5 :': 3.1
`
`0.3 e-. G.0
`
`21.8 ± 1.8
`
`22.3 ± 2.3
`
`2.0::t4.3
`
`Value8 are mean8 _!.. SE; rt, 110. ufexperimeu~~-
`
`lhe ATP-seusilive K+ channels in lhe vasodilai.ion
`evoked by CGRP and levcromakalim.
`H,tfect of" K ; channel antagonists on the ua.sodila.tion
`b_y isoproterenol. When the cortical surface was suf(cid:173)
`fused with mock CSF containing isoprolerenol (l0-6 M),
`the luminal diameter was increased in a concentration(cid:173)
`dependent manner. The vasodilation induced by isopro(cid:173)
`terenol (] 0 6 M), however, was not inhibited by gliben(cid:173)
`clamide (10- 5 M) and charybdotoxin (3 X 10-7 M; Fig. 5).
`
`Changes in r.AMP Prndur.tinn in In Vitrn Experiments
`
`In another series of experiments, we compared the
`effects of CCRP (10- 7 and 10-n M) and levcromakalim
`(10- 6 M) on the stimulation of adenyl cyclase. Signifi(cid:173)
`cant stimulation (P <_ 0.05) was observed with 10-u M
`CGRP bul not with 10-6 M levcromakalim (Fig. 6).
`
`80 A
`40
`
`80 A
`40
`
`t
`CGRP, -7
`
`0
`
`8
`
`~
`
`..
`~ -40
`Q) 80
`E
`«I :a 40
`8i O lff--'4~~tX.:,.,hi__.,ll-
`c::
`t
`!-1!
`CGRP,-7
`0 --40 7--....1----iL--_,..__ ....... _____ _.
`#- ao c
`40
`
`t
`--8
`
`t
`-7
`
`CGRP, logM
`
`0
`
`t
`·9
`
`.. a, 80 B
`
`~ -40
`
`E
`l'O
`:S 40
`C:
`G>
`C) 0
`C
`(U
`s;;, -40
`CJ
`o9. 80 C
`40
`
`0
`
`-40
`
`0
`
`2
`
`4
`
`CGRP,logM
`10 12 14 16
`8
`6
`Time, min
`Fig-. 3. Eflect of suffusion with CGRP no-9 , 10- 8 , and 10- 7 Ml(cid:173)
`containing mock ccrcbrospinal fluid in absence (A) and prcscmce of
`glibendamide (10 GM; Tr) or charyhdotoxin (;; x 10 7 M; C). Valui,.s
`are means + SE from 5 experiments.
`
`-7
`
`log M
`6
`
`t
`CGRP, -7
`
`0
`
`1
`
`2
`
`4
`
`5
`
`--40
`
`3
`Time, min
`r'ig. 2. E ffect of suffusion with CGRP (10-7 Mi-containing mock
`ccrdiro~pinul Jluid in ubGonco (A ) and presence of specific· CGRP
`antibody serum (1:1,000; B) or CGHP-(8-37) (lo-0 .\•l) fragment
`(C). Values are means + SE from 5-6 experiments.
`
`

`

`CGRP AND RA'l' I'lAL ARTEH!OLE
`
`t
`-6
`
`L-CRK,log M
`
`t
`-7
`
`C
`
`.. -40
`-E:
`ao B
`tG
`:S 40
`.£
`8> 0
`CG fl -40 -1,. __ _ . __ ~ __ _ . ._ __ ..__L_-C_R_K_.,,•._0_9__.M 1
`'if. ao C
`
`40
`
`0
`
`-40
`
`I
`-7
`
`0
`
`2
`
`I
`-6
`
`4
`6
`Time, min
`
`L-CRK, log M
`
`8
`
`10
`
`Fig. 1. Effoct of r,uffuflion with lcvcl'Omokolim (L-CRK, 10-7 and
`10 6 M)-containing mock cerebrospinal Jluid in ah;;ence (A) and
`presence of glilwnr.l!imicfo (10-0 M; R) or chorybdotoxin (3 X 10 7 M;
`Cl. Values are means - SE from 4 experiments.
`
`When the pial arteriole was incubated in the depolariz(cid:173)
`ing physiological salt solution (60 mM K- ) containing
`10-G M CGRP, the CGRP-induced increase in cAMP
`production vvas not altered (data not shown).
`
`80 A
`40
`
`0
`
`.. ~-40
`
`~ 80 B
`«I
`:S 40
`C
`(II 0
`Ol
`C
`(V
`.c-40
`(,)
`# 80
`
`40
`
`0
`
`-40
`
`I
`ISP,-6
`
`t
`ISP,-6
`
`t
`ISP, -6
`
`0
`
`1
`
`log M
`5
`
`4
`
`3
`2
`Time, min
`Fig. 5. Effect of suffusion with isoprntl'rP.nol (TRP, ,o-r; M ) inr.lnclP.cl in
`mock ccrcbro~pinu l fluid without (A) ond with pretr eatm ent with
`glibenclamide (10-'' M; B ) or charybdotoxin (3 X HJ ·; M; C). Values
`are mean,; -+: SE from 7 cxpcri men ts.
`
`L-CRK
`CGRP
`Fig. 6. Effects of CORP (10-1; M, left ) anrl levcromakalim (L-CRK,
`10-6 M, ri{[ht) on adenyl cyclase activily and inhibition by CGRP
`antibody serum (1:1,000) and CGRP-(8- 37) fragment (1Q- 6 M) of
`CGRP oo-6 Ml-induced stimulation of cAM I-' production in ml. pial
`arte1-ioles. Open bars, control; filled bars, CGRP or L-CRK: 8tippled
`barn, +CGRP anti,;;erum; hatched bars, +CCRP .(8 37). Values ar c
`means + SE from 4-5 cxperimentH. • l' <. 0.0/5 vs. control; 0 P < 0.05
`vs. CGRP alone.
`
`Ef{P.cfa of' COHP nntihnrfy .'u>.mm and CURP-(8-37)
`fi-agment. CGRP (10- 7 and 10 ° M) caused a significant
`increase in ckviP production in the pial arterioles from
`19.9 + 2.8 (basal level) to 29.5 ± 3.5 and 37.8 .±.. 5.1
`fmol ·mm 2 <JO min - 1, respectively (P < 0.05). The
`increase in cAMP production induced by CGRP (1 o-6 1\.I)
`was significantly inhibited by pretreatment ,,vith CGRP
`antibody serum (1:1,000; P < 0.05 ) or CGl{P-(8-37)
`fragment (10-6 M; P < 0.05), implicating cAl\:IP in the
`CGRP-induced vasodilation of the pial arterioles (Fig. 6).
`Effect nl K ' r.h.rm.nd nn.tngnnist.c; nn. cA MP production .
`The CGRP-inducet.l i11crease in <.AlvIP levels was signifi(cid:173)
`cantly inhibited by churybdot uxin (3 x 10-7 M) as well
`as by glibenclamide uo- 5 M; Fig. 7). However, the
`increase in cAMP production induced by isoproterenol
`(lO· 6 M) was not blocked by glibenclamide (10-~ M) or
`by charybdotoxin (3 X 10 · 7 M; Fig. 7).
`
`DISCUSSION
`
`The major fin<lingt,; of this study in the rat pial
`arterioles arc as follows: 1) the CGRP receptors mediat(cid:173)
`ing vasodilation and stimulation of cAMP accumula-
`
`a
`
`Cl)-
`
`40
`~
`~-~
`g~ 30
`(I) 't
`.!!!E
`~ii:
`u:i
`_c[
`>U
`,, _
`;j 10
`<
`
`20
`
`0
`
`ISP
`CGRP
`l◄'ig. 7. Effects of K- channr.l blockers gl ibe ncla mide (10 ., M) and
`charybdutuxin (3 X 10··7 M ) o n CCRP(lO 6 M, left)- nnd iRoprotere no]
`(ISP, 10 "M, righ t)-induced slimula!.ion of adenyl cyclase in rat pial
`:wt c rinlc., Open b8t'S, control; Jilled lmrs, CGRP ur ISP; stippled bars,
`I glibenclamide; hatched bars, I charybdotoxin. Vnlues m·e n1eans ::t
`s1,; from 4-5 experiments. " f' < 0.05 vs. control: "P < 0.05 vs. CCiRP
`only.
`
`

`

`CCRP AND RAT PU.L ARTERIOLE
`
`HJ21
`
`tion in response to CGRP belonged to the CGRP1
`receptor subtype, 2} vasodilation and accumulation of
`intracellular cAMP in response to CGRP were mark(cid:173)
`edly inhibited by the K+ channel blockers, and 3)
`isuµrolerenol-induced stimulation of aden_yl cyclase
`activity and vasodilation was not inhibited by the K+
`channel blockers. In these experiments, we employed
`glibenclamide (an ATP-sensitive K I channel blocker)
`(:10) and charyhdotoxin (a large-conductance Ca2 +(cid:173)
`activated K- channel blocker) (11) to characterize a
`causative relationship between K+ channel activation
`awl cAMP accumulaLion in response Lo CGRP. In our
`experiments, to ensure that identical arterioles are
`s:rn1plerl, the pin I nrterioles oft.he <'.en~h1·nl sndi:ir.e thnt
`were used for in vivo experiments were dissected and
`utilized.
`Our results showed that arterial diameter and
`adenyl cyclase activity of the pial arterioles were
`increased by CGRP. These CGRP effects were signifi(cid:173)
`cantly antagonized under suffusion with immuno(cid:173)
`reactive specific antibody serum for CGRP and a COOR(cid:173)
`terminal fragment CGRP-(8-37) CGRP, receptor
`antagonist (6). These results indicate that the CORP
`receptors that arc present on the rat pial arterioles and
`mediate vasodilation and cAMP production appear to
`be of the CGRP1 subtype.
`Recently, much evidence suggests that activation of
`ATP-RPnRitivP K+ channPlR anrl Ca2- -ar.tivatPrl K- ch:rn(cid:173)
`nels exerts an important role in vasodilation (24, 26).
`Nevertheless there is some controversy over whether
`the K+ channel openers exert relaxation in the rat
`cerebral vasculature. Mccarron et al. (19) and McPher(cid:173)
`son and Stork (21) reported that K · channel openers
`did not relax the small-resistance arteries from the rat
`cerebral vessels, whereas Nagao et al. (23) demon(cid:173)
`t-iLraLeu crumahalim-iuuuceu relaxaLiun orthe raL posLe(cid:173)
`rior cerebellar artery. We have shown that levcromaka(cid:173)
`lim exerted a strong vasodilator effect, which was
`markedly inhibited not only by glibenclamide but also
`by charybdotoxin, suggesting that the vasorelaxation
`to levcromakalim appears to result from the presence
`and activation of the ATP-sensitive K channels and
`the Ca2 1 -activated K 1
`· channels in the pial arterioles.
`Sovoral difforonL K + channels were demonstrated to be
`responsible for the. actions of K + channel openers in
`various vascular beds (10, 15, 32). Furthermore it is
`known that, in vivo, the pressure drop is larger and the
`regulatory processes are more important in the micro(cid:173)
`vasculature than in large cerebral arteries (22). There(cid:173)
`fore the large regional difference in the responses of
`cerebral arteries might be very dependent on the
`situation of the tissues, for example, in vivo vs. in vitro
`or different preexisLing condiLions (e.g., depolari:11at.ion
`or contracting state).
`WP havP shown t.hat thP C(;l{P-inrln<'.Prl vasorlilation
`was inhibited by not only glibenclamide but also charyb(cid:173)
`dotoxin, as was the levcromakalim-induced vasodila(cid:173)
`tion. Glibenclamide and charybdotoxin showed no addi(cid:173)
`tive action on the CGRP-induced vasodilation. It is not
`known how ATP-sensitive K- channels and large(cid:173)
`conductance Ca2 - -activated K+ channels are activated
`
`by CGRP in the pial arterioles. Carl et al. (4) demon(cid:173)
`strated that, in cell-attached patches from canine colon
`cells, levcromakalim increased the open probability of
`large-conductance Ca2 + -activated K 1 channels, which
`were prevented by glibenclamide and tetraethylammu(cid:173)
`nium. Thus further study is required to identify electro(cid:173)
`physiologically whether the K+ channels activated by
`CGRP in the pial arteries share the properties of
`ATP-sensitive K + channels and Ca2 - -regulated K- chan(cid:173)
`nels. Recently, Trezise and Weston (33) compared the
`relaxant and hyperpolarizing actions of CGRP with
`those of BRL-38227 (levcromakalim) iu Lhe raLLiL
`basilar arteries. They reported that CGRP consistently
`P.vokr.d a glibenclamidr.-inhibitrible hypr.rpolarizrition ,
`though it was less than that evoked by BRL-38227.
`Vasorelaxation induced by CGRP has been proposed
`to be mediated by two interesting pharmacological
`actions: 1) stimulation of adenyl cyclase activity with a
`subsequent accumulation of intracellular cAMP (7, 9)
`and 2) activation of the K- channels and subsequent
`hyperpolarization ('25, :~5). The role of the K + channels
`was reported in the vascular response to several endog(cid:173)
`enous vasodilators (1, 3}.
`We have shown that CGRP significantly stimulated
`the adenyl cyclase activity with subsequent accumula(cid:173)
`tion of intracellular cAMP in the pial arterioles. This
`CGRP effect on cAMP production was markedly inhib(cid:173)
`itP-rl hy glihPnclarnirfo :rnri cha.ryhrlotoxin. ThPRP find(cid:173)
`ings strongly support a role for the ATP-sensitive K+
`channels and the large-conductance Ca2--activated K+
`channels in the CGRP receptor-coupled cAMP produc(cid:173)
`tion and vasorelaxation. In support of this conclusion is
`Lhe recent report (31) that a hypcrpolarization-acti(cid:173)
`vated K I conductance of the cell membrane of Parame(cid:173)
`cium directly stimulates adenyl cyclase activity as a
`carrier or ~he K+ re::;Liug cumlucLam:e. However, iL has
`not been reported whether the K + conductance-coupled
`cAM !-'-producing system exists in the smooth muscle
`cells. We have shown that CGRP-induced stimulation
`of adenyl cyclase activity was not affected by levcro(cid:173)
`makalim , a hyperpolarizing agent, or by depolarizing
`) . It is likely that
`physiological salL solution (60 mM K 1
`the activation of adenyl cyclase is not influenced by a
`change in mcmbrnno potential but is linked to an
`adjacent K+ channel by an unknown mechanism. It is
`not clear how CGRP increases the K' conductance in
`the pial arterioles and whether the hvo K ' channels act
`independently or cooperatively.
`Recently, Nelson et al. ( 25) showed that CGRP acti(cid:173)
`vates an ATP-sensitive K+ channel in single smootl1
`muscle cells from rabbit mesenteric artery, and Ed(cid:173)
`wards et al. (9) suggested that CGRP receptors in
`va~cular ~muuU1 mucde are couµled Lo adeuyl cyclase.
`Thus it is considered that activation of adenyl cyclase is
`dosPly linkPrl to thP K channPls in vaRrnlar smooth
`muscle cells. However, we do not know whether the K+
`channel blockers such as ghbenclamide and charybdo(cid:173)
`toxin directly block the CGRP receptors. Additional in
`vitro studies are needed to identify a causative relation(cid:173)
`ship between the inhibition of adenyl cyclase activity
`and CGRP receptors by these K + channel blockers,
`
`

`

`H322
`
`CGRP AND RAT PIAL ARTERIOLE
`
`because Trezise and Weston (33) demonstrated ihaL
`CCRP and 1evcrornaka1im are capable of opening glib(cid:173)
`enclamide-senRitive K+ rhannels in rahhit hrisil:w rir(cid:173)
`tery. Otherwise, it can be considered that CGRP-evoked
`rcluxution is almost dependent on the adenylate cy(cid:173)
`clase activation, whereas levcromakalim-induced relax(cid:173)
`ation is certainly a conRequence of the opening of Ki·
`channels.
`On t.he other hand, a number of results regarding
`cAMP-dependent K- channel activation have been
`demonstrated in cultured smooth muscle cells of rat
`aorta (28), in Drosophila. larval muscle (5), and in rat
`basilar artery (16). We examined the responses of the
`pial arkriole:o Lu i::;oproLereuol (B-a<lrenoceptor activa(cid:173)
`tor), which activates adcnyl cyclase via G protein. As
`mentioned above, neither isoproterenol-induced vasodi(cid:173)
`lation nor isoproterenol-induced increase in cAMP pro(cid:173)
`duction was aflected by the K+ channel antagonists
`glibenclamide and charybdotoxin. Hiramatsu and co(cid:173)
`workers (13) reported the suppression by charybdo(cid:173)
`toxin of the isoproterenol-induced relaxation of the
`guinea pig tracheal smooth muscle, and they postu(cid:173)
`lated that isoprotcrenol-induced relaxation occurs at
`least in part through opening of charybdotoxin-sensi(cid:173)
`tive K+ channels by means of a cAMP-dependent
`channel modulation. However, the preRent st.miy with
`rat pial arterioles, in contrast, showed lack of effects of
`glibcnclumidc und c:hurybdotoxin on the isoproterenol(cid:173)
`induced relaxation, thereby suggesting a lack ofinvolve(cid:173)
`ment of the hyperpolarization of cell membrane by
`stimulation of the ATP-sensitive K+ channels and
`Ca2+-activated K+ channels. We could not provide
`direct evidence regarding the involvement of cAMP(cid:173)
`depcndent K+ channel activation without patch-clamp
`techniques.
`Taken together, it is suggested that the CGRP1
`ret:eplurs, when activated by CGRP, are coupled to
`adcnyl cyclase activation and opening ofK- channels in
`the pial arter.ioles.
`
`Address for reprint requests: K. W. Hong, Dept. of Pharmacology,
`College of Medicine, Pusan National University, A.mi-dong 1-Ga,
`Seo-gu, Pusan 602-739, Korea.
`lfo1:eived 24 January 1995; a~~eµLeu iu final form 13 June 1995.
`
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