Sep 14, 2017 - monitored with a Dual-channel Lumi-aggregometer (model. 400, Chronolog Corporation, Chicago, IL, U.S.A.) using. 0.45 ml aliquots of ...
BIOCHEMICAL SOCIETY TRANSACTIONS
1. Willis, A. L. ( 1978) Hutidbook o] Experimental I’hmrmcicology (Vane. J. R. & Ferreira, S . H.. eds.). vol. 50/1. pp. 138-205. Springer-Verlag. Berlin. Heidelberg. New York
Received I4 September I Y X Y
A comparative study of the effects of local anaesthetics on platelet aggregation ANWAR H. GlLANl and SHEIKH A. SAEED Department #f’~-’hurmuco~o&y, 7’heAgu Khun University Medical College, Kuruchi 74800, Pakistan A variety of drugs that are generally thought to modify biological membrane action may affect platelet function [ 11. Phospholipase A? is a membrane-bound enzyme, and plays a key role in the release of arachidonic acid from platelet membrane phospholipids [ 21. The arachidonic acid thus released is converted by platelet cyclo-oxygenase and lipoxygenase pathways to prostaglandin endoperoxides, thromboxane A: and hydroperoxyeicosatetraenoic acid, resulting in platelet aggregation. In the present investigation we sought to evaluate the effect of local anaesthetic agents (thought to act by stabilizing membrane) o n platelet aggregation induced by various aggregating agents. Blood was taken by venepuncture from normal human volunteers reported t o be free of medication for 1 week. Blood samples were mixed with 3.8% (w/v) sodium citrate solution (9: 1 ) and centrifuged at 260 g for 15 min at 20°C to obtain platelet-rich plasma. The remaining blood sample was centrifuged at 1200 g for 10 min to obtain platelet-poor plasma. Platelet count was determined by phase-contrast microscopy and all aggregation studies were carried out at 37°C with platelet-rich plasma having platelet counts between 2.5 and 3.0 x 1Ox/ml of plasma. Aggregation was monitored with a Dual-channel Lumi-aggregometer (model 400, Chronolog Corporation, Chicago, IL, U.S.A.) using 0.45 ml aliquots of platelet-rich plasma. The final volume was made up to 5 ml with sodium chloride (0.9’/0, w/v) or test drug and incubated for 1 min before challenge with the Abbreviation used: lC5,,,concentration producing 50”% inhibition of platelet aggregation.
aggregating agent. Aggregation was induced with ADP (2.2 pM), sodium arachidonate ( 1.7 mM) o r platelet-activating
factor (0.8 p ~ )The . resulting aggregation was recorded with a Lumi-aggregometer and expressed as percentage inhibition compared with control at 4 min after challcngc. All drugs were. tested at three to six different concentrations in duplicate. Statistical differences between control and drug-trcated platelet preparations were determined by Student‘s r-test. Table 1 summarizes the effects of various local anaesthetic drugs on platelet aggregation induced by ADP. arachidonic acid and platelet-activating factor. All local anaesthetic agents studied inhibited platelet-activating-factor-induced aggregation in a concentration-related manner. The order o f potency, in increasing fashion, was benzocaine, lignocaine, benoxinate, tetracaine, alcaine and procaine with concentrations producing 50% inhibition of platelet aggregation ( lC5,,j of 240, 235, 170, 100, 80 and 78 p ~ respectively. , Only alcaine, benoxinate and procaine were found t o have weak inhibitory effects against arachidonic acid-induced platelet aggregation, with IC,,, values of 250, 200. and 180 p ~ . respectively. Similarly ADP-induced aggregation was inhibited by procaine and tetracaine only. Our data demonstrated that all local anaesthetic drugs studied exert a strong inhibitory effect against plateletactivating-factor-induced aggregation. Previously, general anaesthetics have been reported t o have an inhibitory effect on platelet aggregation induced by ADP (31. Furthermore. we have recently described a selective, inhihitory effect of anti-arrhythmic drugs against platelet-activating-factorinduced aggregation [4].Taken together, these findings suggest that the inhibitory effect of local anaesthetic and anti-arrhythmic drugs on platelet aggregation may be related to their ability to stabilize or penetrate the lipid monolayer in the plasma membrane.
Table I . Inhibition of human pfatefetaggregation by locaf anaesthetics All values are mean sf^.^.^. of three to six experiments. Assay conditions were as described in the text. Statistical significance was assessed by Student’s t test. Abbreviation: mi., not inhibited up to 500 PM. IC,,, ( w ) Arachidonic
Drug Alcaine Benoxinate Benzocaine Lignocaine Procaine Tetracaine
Aggregating agent.. . ADP n.i. n.i.
acid 250 f 8 200 f 9
n.i.
n.i.
n.i.
n.i. 18Oi6
300k 10 182k7
n.i.
Plateletactivating factor 80 k s 170*8 240 k 10 23Sk 10
78iS 100+6
1990
289
632nd MEETING, C O R K Wc t h a n k M r Hazratuddin Khan for technical assistance. 1. Seeman. P. M. ( 1986) Int. Rev. Neurohiol. 9 , 145 2. Longenecker, G. L. (198.5) in The I’lotelers: /’hy.siology crnd /’harmcrcology (Longenecker. G. L.. ed.). pp. I S9- 185. Academic Press, Inc., San Diego, New York
3. Ueda, 1. ( 1 07 I ) Anrresrhesiologv 34,405-408 4. Gilani. A. H. & Saeed. S . A. ( 1989) Hiochem. Soc. Tmns. 1 7 . 9 0 2
Received 14 September 1 980
Comparison of the rate of occupancy of receptors by anti-cholinergic drugs A N WA R-U L-H ASSA N GI LAN I* I~epurtmcntof’l’hurinucokgy, University of’.Yydney,.Sydney, N S W ZM. Airstruliu T h e physiological role of acetylcholine is mediated through activation o f either muscarinic or nicotinic receptors. T h c muscarinic responses to acetylcholine that are blockcd by atropine are further subdivided into two types based o n the antagonist affinities for muscarinic receptors in differcnt tissues [ 1 I. Receptors which are blocked by low concentrations o f pirenzepine, an anti-musearinie drug, arc called as neural ( M , )and those which are insensitive t o low concentrations of pirenzepine peripheral (M,) receptors. Peripheral muscarinic receptors are further subdivided into cardiac and smooth muscle receptors as himbacine, a plant-derived alkaloid, exhibits at least 10-fold higher affinity for cardiac muscarinic receptors [ 21. We have recently proposcd that himbacine may be a pharmacological tool for studies involving heterogeneity o f muscarinic receptors in cardiac and smooth muscle [ 3 ] . However, the rate o f occupancy o f muscarinic receptors for himbacine is yet t o be determined. In this investigation, the onset of action o f himbacine was studied and compared with that of atropinc and/or homat ro pi ne. T h e onset o f antagonism of himbaeine and atropine was studied in isolated guinea-pig ileum and atria. In ileal experiments, when responses were stable to acetylcholine the bathing fluid was replaced with Tyrode’s solution containing an antagonist and isotonic responses to aeetylcholinc were recorded on Grass model 7 9 polygraph at constant time intervals until constant responses were obtained. T h c responses were plotted against time as shown in Fig. 1( u ) and the time required for equilibrium was estimated as dcscribed by Thorn & Waude [4]. T h e method of Madden 8: Mitchelson [ S ] was followed for the study of the onset o f antagonism by himbacine. atropine and homatropinc of negative inotropic responses to carbachol in electrically stimulated left atria o f guinea-pigs ( 3 ms; 2.5 Hz). Responses were measured via a force-displacement transducer (FT03) coupled t o a Grass model 7 9 polygraph. A carbachol concentration of 0.1-0.3 PM produced 70-90% inhibition o f the control force o f contraction of the atria usually within 3 min. T h e antagonist was then added t o the bath as shown in Fig. l ( h ) and the onset of antagonism, i.e. the time taken for carbachol responses to be reversed by 50%. was calculated by regression analysis. T h e time course of development of antagonism o f aeetylcholine by himbacine and atropine was studied in five ideal preparations and the results are shown in Fig. 1 ( u ) . Both himbaeine ( 1 P M ) and atropine (10 nM) at equi-effective concentrations commenced antagonizing the contractile response to acetylcholine immediately after contact with the tissue, but the time required to achieve maximum antagonism varied between the drugs. In three out of five experiments, himbacine exerted its maximum antagonistic effect almost immediately, whereas in the other two preparations it took *Present address: Department of Pharmacology, The Aga Khan University Medical College, Karachi 74800, Pakistan.
Vol. 18
2.5-
.
2.0. 30 0
1.5.
0
Atropine
.
0
0
1.0.
1.5. Himbacine 0
.
1.0.
0.51 7
0
5
10
1’5
20
25
Time (mini
Homatrooine
-
I’ Atropine
1 min
Fig. 1. Ai~tugoi~orlismoJ hiinhuciric and utropinr in isoluted giiirierr-pig ilcwrn u i i d utriu
( u ) Comparison of the onset of action of hirnbacine and atropine in guinea-pig ileum. T h e symbols represent five different preparations. Ordinate: response to acetylcholine relative to that reached at equilibrium; abscissa: time after replacing bathing fluid with one containing antagonist. ( h ) Representative traces showing reversal o f carbachol responses by himbacine, hornatropine and atropine in guinea-pig left atria driven electrically. At the concentrations shown for the different antagonists, the magnitude of the effects, in terms of agonist dose-ratios, were comparable. T h e symbols represent times when drugs were added to the tissue bath.
