33

to caffeine and acetylcholine in guinea-pig coronary endsthe1ia.I cells Guifa Chcn and Donald

Received

6 Jmuary

IYY?. revised MS received

W.

7 August

Cheung

IYYL?. :tccrptcd

IX August

WY2

Membrane potential changes in cndothclial ccll~ in tcsponse to caffcinc and acctylcholinc (ACh) were rccordcd with microclcctrodcs from an intact cndothclium preparation from the guinea-pig coronary artcry. Caffcinc induced ;I transient hypcrp(}i~riz~lti~n of the mcmbrenc in a concentration-dcpcildcnl manner. The i~ypcrpoi~riz~ti(~nwas inhibited by removal of Ca3+ from the bathing medium and by ryanodinc (20 @MI. It was not affcctcd by ~,4,S-trimctht)xybcIlzoic acid ~-(di~thyl~min~~) octyl cstcr hydrochloride (TMB-8, IO PM) or neomycin 15 mM1. ACh induced a sustained hypcrpolarization in cndotheiial cells. At concentrations that caused no significant cffccts on the caffcinc rcsponsc, TMB-8 and neomycin inhihitcd hyporpolarization induced by ACh. Ryanodinc did not inhibit the response to ACh. The ACh-induced hypcrpolarization was also inhibited by caffeine in 8 c~nc~ntr~ti(~n-dcpcnd~nt m~nncr. Results from the present study suggest that hyp~rpol~riz~tions induced by caffeine and ACh arc mcdiatcd by scparalc Ca’ ’ pools.

Endothclial cells: Cnffcinc: Ca” ’

1. Introduction Activation of muscarinic receptors by a~~tylcllolin~ (ACh) increases cytosolic free Cal+ in c~d~~thclial cells by releasing Caz + from intracellular stores and promoting influx of cxtracellular Ca’+ (Danthuluri ct al., IYSgf. The increased Ca’+ stimulates a Ca’+-dcpcndent K+ outward current, resulting in hypcrpolarization of the endothclial cells (Bussc et al., lY88). In cndothclial cells from the guinea-pig coronary artcry, the ACh-induced hyp~rpolarization is depressed by the K+ channel blockers charybdotoxin, tctracthylammonium, and 4-aminopyridinc (Chcn and Chcung, 1092). The hyperpolarization response is not mcdiatcd by ~ndothclium-dcrivcd relaxing factor, cGMP, a cyclooxygenase product cr stimulation of clcctrogcnic Na+, K+ pumping (Chen and Chcurrg, lYY2). Recent studies in endothclial and other cc11systems indicate that inositol l,4,~-triphosphat~ UP31 may play an important role in the signal transduction process upon receptor activation (Bcrridgc, 1001; Jacob, 1091). Thus IP, may induce release of Ca’+ from a specific IP~-sensitive pool and influx of cxtcrnai Ca”. Cal+ released from the II’,-scnsitivc pool may trigger a sccondary release from a separate IP3-inscnsitivc internal

f-y+-

pool fCa’+-induced Cal + rclcasc) (Bcrridgc, lYYl I. The ryanodine receptor functions as the 01” ’ release channel in this caffci~e-sensitive Ca?+ pool (McPherson ct al., 1YYl I. Separate IP,-gatcd channels dnd tyanodinc rcccptors/ channels have rcccntly been idontificd from electrophysiological stud& on cndoplasmic reticulum vcsiclcs from canine ~~r~bcllurn incorporated into planar hilayers (Bczprozvanny ct rtl.. IYYl). Par!icipation of t!rc IP3-scnsitivc Ca’+ pool in agotwist-induced rcsponscs in ~ndoth~li~ll cells has been well documented. In saponin-pcrmcabilizcd cells, IP3 inducts a rapid and large rclcssc of Ca2+ constituting 74% of the non-mitochondrial fraction I$ tho Ca”” store t&cay et al., IYgYI, Ca’* in the IP~-sensitive pool can hc mobilized by agonists such as bradykinin which stimulates IP, production (Lamhcrt ct al., IYXclI. The cffcct of caffcinc, however, has not been characterized in cndoth~lial cells. In the prcscnt study, WC ~xalnin~d the roles of the IP,-scnsitivo and Cilffcillc-SCJISitiVC Ca’.’ pools in the membrane liypcrpolarizatioll IXsponsc to ACh and caffcinc in an intact cndothclium prcp~tration from guinea-pig ~~~ro~la~artery.

2.

Mutcrirls and

methods

Thu cxpcrimcntal procsdurcs wcrc similar to those previously dcscribcd (Chcn and Chcung, lYY2). The &cumflcx coronary artcry was disscctcd out from

?OO-410 g in weight and cut open. The intima layer ~vas gently separated from the rest of the artcp with a pair of fine forceps and pinned to the bott~?~~ of an organ bath. The p~~p~~~ilti~~ WIS ~_l~rluscJ at I rate of 2 ml/min with physiological solution bubbled with 05% 0, and SC/CCO,. and containing (in mM): NaCl 120, NaHCCP, 25, glucose 31, KC1 5, CaUt, 2.5. N~~~~Q~ 1. and MgSO, 1. In nominally @‘-free solutions, CaCI, was omitted. Class micropipettcs filled with 3 M KCI were used for intracellular recordings. M~~~b~~~ne~ote~ti~l~ were measured with a Dagan ~7(~) i~traceltutar preamplifier (Dagan Corp.. Minneapdis. MN). The cndotbcliai cells wcrc impaled from the luminal surface. The fot~owi~g chemicafs wcrc used in the study: acctykholinc chloride (Sigma). caffeine (BDHI, ncomycin sulphate (BDHJ, 3,4,5trimethoxybenzoic acid ~-(d~~tby~amino) octyl ester hydrochloride (TM&8, Sigma). ~anudin~ (Calbiochem). Preliminav studies showed that the responses to ACh and caffcinc were rcproduciblc if a washout pcriod of IS min was allowed between applications. Thcrcfore in alf experiments a washout period of at least 15 min was imposed hctwcen applications. The effects of various agents on the responses to ACh and caffeine were compared before and after trcatmcnt on the same preparation. Ryanodinc and TMB-8 were added 10 min, and neomycin 5 min, to the perfusing solution bcforc their effects on the hyperpolarization response to ACh and caffeine were tcstcd. A11 dats were presented as means + S.E.M. n denotes the number of preparations. Student’s paired t-test was used for statistical analysis. P G 0.05 was the specified level of sign~fi~ance.

guinea-pips

10 m&f -\

../-------‘,./ri/

r/

20 mV

Lset 30

Fig. I. M~rnl~~~n~ raponsc of the ~nd~~hcl~l ccl1 to incr~~s~n~ concentrations of caffkw. A washout period of al least IS min was aliowcd hcrwoen each applicahn. All recordings wcrc made in the 531716crll.

The hyp~r~otari~tion rcsponsc to caffeine is sensitive to removal of CL?+ from the perfusing solution. The hypcrpolarization decreased with each successive ~pplj~t~on of Eaffeinc and the response was aholishcd after six to seven appfications (fig. 21.

3. Results

The membrane response to caffcinc is concentration-dependent. A small transient hypcrpolarization could he elicited at a threshold concentration of 0.5 mM (fig. 11. The hyperpolarization increased progressively with increasing concentrations of caffeine, up to a maximum of 20.2 + 1.3 mV (n = 8) at 50 mM caffeine. As the hyperpolarization increased, the duration of the response decreased so that at 50 mM, the response became a sharp peak. At concentrations higher than 5 mM, a slow sustained depolarization appeared following the hypcrpolarization (fig. 1). To avoid possibk effects of increased osmolality, a caffeine conc~ntrati~?n of 5 mM was used in ail subscquent experimonts although similar effects were observed with 50 mM caffcinc.

The hyperpolarization response to caffcinc was abolished by ryanodine (20 @MI. The hyperpolarization to 5 mM caffeine was 17.5 C 1.0 mV Cn = 4) (fig. 3). This was significantly reduced to 0.6 it: 0.4 rnV in the prescncc of tyanodine (table 1). There was no significant change in the resting membrane potential with ryanodinc treatment (control = -44.4 f 0.5 mV; ryanodine = -44.6 k 0.4 mV; n = 4). The caffeine response was not affected by the intracellular Ca2+ antagonist TMB-8 (10 PM) (control = 16.0 k 0.7 mV; TMB-8 = 16.1 f 0.8; n = 5) (fig. 3). TMB-8 also caused no significant change in the resting membrane potential of the endothelial cells (control = - 43.0 _I 0.6 mV; TMB-8 = -42.3 jlO.7 mV; n = 51. Neomycin (5 mM) caused significant depolarization of the memb~ne (control = - 41.2 f 3.8 mV; neomycin = - 32.3 f 1,O mV; P < 0.05; n = 11). However, it had no cffcct on the amplitude of the hypcrpolarization rcsponsc to cef-

feint fcontroi = 17.0 f 0.7 mV; neomycin = 16.9 t 1.2 mV; n = 6) (fig. 3).

The endotheliaf cells hypcrpolarized ii-1response to

V

-1

20 mV

30 see Fig. 2. CI++ Jcpendencc of the hyperpol;lrizatiu,n rcsponsc to atffeine. When CaCIz wilS omitted from the physioltrgical solution. tbo hy~rp~~~riza:j~n to caffeine decreased pngressively with ach successive application uf ihe agent. A washout period of et kitst IS min was allowed between caffeine upptications. ~(~ntinu(su~ r~curding from thr: same cell.

ACh in a concentration-dependent manner (Chen and Cheung, 19921. The response was characterized by an initia1 drop in membrane potential followed by gradual repolarization (fig. 4:. At higher concentrations, the rcpolarization process was slow so that more than W min was required in order for the m~mbr~~ne to return to the resting level (Chen and Cheung, 1992). The ACh rcsponsc was also dcpencient on external Ca’+. In nominally Ca’+-free solutions, ACh elicited only a ~~~ns~~~~lly~~rpolar~~t~on which decreased in ampiitudc with each subsequent application of ACh iChcn and Chcung, 1992k

-JO

-

-40

-

caffeine D t mhi I 5 mM [Ii ton-d.4

I-1 10-q

10“

1o-8

10-6

10-5

Fig. 6. Graph showing the effects of various concentrations of caffeine on the hyperpolarization response to cumulative concentrations of ACh in guinea-pig coronary endotheliai cells. n = 4-h. Ordinate: hype~~larizatio~ fmVf; abscissa:ACh (Ml.

7) (table Fig. 4. Membrane response of the end(~thelial cell to increasing co~c~~tratjons of acetyicituiiii~. A Gashout period of at least 1.5min was allowed between each application. w = wash. Continuous recording from the same cell.

3.5. ~ha~~colo~

of the ACh-induced hyperpolarization

To compare the effects of ryanodine, TMB-8, and neomycin on the hyperpolarization response with caffeine, ACh at a concentration of 0.1 PM that produced a similar degree of hyperpolarization was chosen. At a concentration that produced no effect on caffeine-induced hyperpolarization, TMB-8 (10 FM) completely abolished ACh-induced hypcrpolarization (fig. 5A, n =

L ACta0.i

1). The ACh-induced hyperpolarization ‘was also inhibited significantly by 5 mM of neomycin tcontrol = 23.4 * 1.2 mV; neomycin = 15.0 It 1.3 mV; P < 0.01; n = 8). Only a transient hyperpoiarization remained and the membrane repolarized quickly to the resting potential (fig. SB). Ryanodine (20 PM), which abolished the hyperpolarization to caffeine, caused no significant change in the hyperpolarizatiorf response to ACh (control = 20.7 rf: I.8 mV; ryanodine = 20.8 f I.2 mV; n = 4) (table I).

3.6. Effect

ofcaffeine

on AC%induced

hyperpolarization

The ACh response was inhibited by caffeine in a concentration-dependent manner {fig. 4). The hyperpolarization became more transient and the membrane

I--.4(1

?,‘r‘

j&f

Fig. 5. Effects of TMB-8 and neomycin on the response to ACh (0.1 pM1. (A) AC%induced membrane hypcrpolarization was abolished after Pretreatment of the preparation with TMB-8 (110 FM) for 10 min. (B) The hyperpolarization response to ACh was reduced in amplitude and dura~on in the presence of neomycin (5 mM1. w = wash.

37

Ryanodine inhibits the caffeine response by depicting Cat” from the caffeine-sensitive pool (Hansford and Lakatta, 19873. In the prcscnt study, the majntenancc of the caffeine response requires extracellular Ca”. The decrease in response was gradual, becoming progressively smaller with each subsequent application of caffeine. This would suggest that the caffeine-sensitive pool was repleted with cytosolic Ca’+, which in turn relies on extracellular Ca2+ for replenishment. Caffeine also induced hyperpolar~zation by releasing Ca’+ from a ~anodine-sensitive store in other cell types (Fujii et al., 1985). In higher concentrations, caffeine induced a sustained depolarization in the endothelial cells subsequent to the initial h~rpolari~ation and was not dependent on extracel~urar Ca’+. A similar depolarization, accompanied by an increase in membrane resistence, was also observed in vascular smooth muscle in high concentrations of caffeine (Fujii et al., 1985). Facilitated extrusion of Ca ‘+ from the cytolasm due to stjmulation of a Caz’-dependent ATPase was offered as the explanatjon- WC have not examined the mechanism of caffeine-induced depolarization in the endothelial ceils.

Fig. 7. Inhibitory effects ctf increasing c~~ncentr~lti~n~of eaffeinc on the hyperpolarization response to ACh 10.1 PM). Ctlffeine was added 5 min before ACh in each case. At least 15 min w;is allowed between t&s and the membr~oe had c(lrn~letely repolarized to the resting level when ACh WE applied. w = wash.

repolarized quickly to the resting membrane potential with increasing concentrations of caffeine (fig. 7). Caffeine was atso effective in immediately terminating the ACh response when added in the middle of a sustained hyperpolari~ation.

4. Discussion

Caffeine induces a transient hyperpolarization in coronary endothelial cells in contrast to a sustained response to RCh. The hyperpolarization induced by caffeine, but not by ACh, was effectively blocked by ryanodine. It has now been demonstrated the ryanodine receptor functions as a caffeine-sensitive Ca2* release channel ~~cPherson et al,, 1991; Bezpromanny et al., 19Yl). Ryanodine increases the open provability of the release channel but reduces the unit conductance to Ca 24 by almost 2-fold {Roussc~~i ct al., 19871,

The characteristics of the h~crpo~ari~ation response to ACh are totally different from that to caffeine. In contrast to the transient effect of caffeine, a long lasting byperpol~rization more than 30 min in duration could be elicited by ACh (Chen and Cheung, 1 92). ~yanodine, which effectively abolished the caffeB,I e-induced hyperpo~ari~ation, had no inhibitor cffeet on the response to ACh. Conversefy, TMB-8 and neomycin, the two agents that had no significant cffccts on the caffeine response, inhibited ACh-induced hyperpolari~at~on- TMB-8 has been described as an “intraceltufar Csz* antagonist’ iChiou and ~a~ag~~~ 1975). In general, it reduces the availability of Cal.” within the cell. At the concentration used (10 PM), TMB-8 abolished only the ACh response without affccting the caffeine response. This further supports the suggestion that ACh and caffeine induce Ca’+ release from different pools. Results from the neomycin cxpcrimcnt also lend support to this conclusion. Neomycin inhibits membrane phosphoinositidc metabolism (Schacht, 1976). In the prcscnt study, neomycin was found to have no significant effect on the hypcrpolarizntion induced by caffeine but that induced by ACh was significantly reduced.

Although the hypcrpolarization rcsponscs to caffcinc and AC% could be separated pharm~cologic~lly~

the issue \v;1~ complicated hy the inhibitory action of &nr: on the ACh response. At concentrations that elicit on& a s~~a~~ ~y~~rp~~~arizatj~~n by itself. caffeine mechanism

effect

of action

on AC% response

underlying

this inhibitory

by caffeine

is not known. A recent study on X~~~lf?~l~~ oocytes suggests that caffeine may inhibit liber~t~~~n of Ca’+ by 1P3 (Parker and Ivorra. 10911. Whether the inhibitory action of caffeine

on AC%-induced hyperpolarization is due to a similar ~~~h~~nisrn remains to be determined. In addition, caffeine

is also

known

to have direct

and

indirect

action on many ionic channels. For example, caffeine has been reported !o block voltage-operated Ca)+ ~~?ann~~s(Hughes et al.. IYYO) and affect many other ionic channels including a transient outward K” channel, a transient Cl channel, a Na + channel, and a sustained K’

Results

channel (Akaike

from

the

present

and Sakoshima,

IYXY).

study suggest that

the

hype~olarizatil~n response to caffeine and ACh may be mediated by two separate pools of Ca”‘. The hypcrpolarization rcsponsc to ACh can be inhibited by neomycin, TMB-8. and caffeine and is probably mediated by 1P3. In contrast, the response to caffeine can be sctcctiveiy inhibited by ryanodinc. Both Ca’+ pools require replenishment from extracellular sources.

This work was supported hy the Heart & Stroke Foundation of Ontario. Gc’. is a research fellow of the lleart Kr Stroke Foundation of Canada.

References Akaike. N. and J. Sakoshima. IYHY. Caffeine affects four different ionic currrnis in the bull-frog sympathetic ncuronc. J. Physiot. (L4mdonI 41’_. ‘11 __ .

ilcrridge, M.J.. IYYI. Cytoplasmic calcium oscillations: a two pool model. Cell Calcium 17. 6.3. Ucqm,manny. I.. J. Watras and B. Ehriich. IYYI. Bell-shaped calrium-rssponsc curves of Ins{ t,4.SfP1- and calcium-~ate~i channels from endoplasrnic reticulum ctf cerebellum. Nature .%I. 751. Busse. R.. H. Fichtner. A. Liickhoff and M. Kohlhardt. IYXX. Hypcrptrlarizatitrn and increased free calcium in acctylcholine-stimulated endothrlial cells. Am. J. Physiol. 25.5, I1965 Chen. G. and D.W. Cheung. 1992. C~racterizati(~n of the acetylcholine-induced membrane hyperpolarization in endotheiial cells, Circ. Rcs. 70, 257. C’hiou. C.Y. and M.H. M;dagodi. 1975, Studies on the mechanism of action of a new calcium antagonist TMB-X in smooth and skeletal muscle. Br. J. Pharmacol. 53. 170. Danthuluri. N.. M. Cybulsky and T. Brock, IYXX. ACh-induced calcium transients in primary cultures of rahhit anrtic endothelial cells. Am. J. Physioi. 255. HlS4Y. Frcay. A.. A. Johns. D.J. Adams. LJ. Ryan and C. Van Breemcn, ISXO. Bradykinin and inositol l.4.S-triphl~sp~t~-st~mulated calcium relcase from intracellular stores in cultured hovine endothelial cells. Pfliigcrs Arch. 414. 377. Fujii. K.. Il. Miyahara and H. Suzuki, IOXS, Comparison of the effects of caffeine and procaine cm nttradrenergic transmission in the guinea-pig mescntcric artery. Br. J. Pharmacol. X4, 675. Hansford. R.G and E.G. Lakatta. 10x7, Ryanodine releases calcium from sarcoplasmic reticulum in calcium-tolerant rat cardiac my. ttcytes. J. Physic& London) 3Y0, 453. Htighes. AD.. S. Hering and T.B. Rolton, IYYtf. The action of caffeine on inward barium current through voltage-dependent calcium channels in single rabbit ear artery cells, Pfltigers Arch, 416, 462, Jacob, R., IYYI. Calcium ~~seill~~ti~~ns in end~lthc~ial cells, Ceil Calcium 12. 127. Lambert. T.L.. R.S. Kent and R. Whorton. lYX6. Bradykinin stimulation of inositn! polyphosphate production in porcine aortic cndl, 152X8. McPherson. P.S.. Y. Kim, 11. Valdivia. C. Knudson. ii. Takekura, C. Franzini-Armstrong. R. Coronado ;md K.P. Campbell, 1091, The brain ryanodine receptor: a caffeine-sensitive calcium release channel. Neuron 7, 17. Parker, i. and I. Ivorra, I‘JYl, Caffeine inhibits inositol trisphosphate-mediated lihuratbn of intracellular calcium in ,%tro/>u,r oocytes. J. Physiol. ILrmdon) 433. 220. Rousseau. E., J.S. Smith and G. Meissnrr, 19X7, Ryanodinc modifies conductance and gating behavior of single Ca2 ’ release channel, Am. J. Physiol. 253, C314. Schacht. J.. 1976. Inhibition by neomycin of polyphosphoinositidc turnover in subcellular fractions of guinea-pig cerebral cortex in vitro. J. Neurochem. 27. I 119,

Pharmacological distinction of the hyperpolarization response to caffeine and acetylcholine in guinea-pig coronary endothelial cells.

Membrane potential changes in endothelial cells in response to caffeine and acetylcholine (ACh) were recorded with microelectrodes from an intact endo...
703KB Sizes 0 Downloads 0 Views