35P
Medical Research Society
lnitially I developed and validated an assay system capable of
measuring PAP activities in human needle liver biopsies. nKn.
Using gel filtration and anion-exchange chromatography, I have provided physical evidence for 2 distinct forms of PAP in rat liver: one
present in the cytosol and micmsomes. sensitive to inhibition by N-
ethylmaleimide (NEM). with a role in higlyceride and phospholipid
metabolism, and another present in the plasma membrane, insensitive to NEM. with a putative role in cell-signalling.
I have investigated the hypothesis chat susceptibility to alcoholic
fatly liver (AFL) is determined by inter-individual variation in the
activity of the metabolic form of PAP by measuring PAP activities
in needle liver biopsies from 42 alcoholics and 6 pts with primary
biliary cirrhosis (PBC) and in wedge biopsies from 6 'normal" pts
undergoing routine cholecystectomy. Steatosis was 'scored' on
coded slides from 0-3. NEM-sensitlve activity was higher in 'alcoholic' biopsies scoring 3 (3.25k0.4 unitslmg. n=10) than those
scoringO(l.Zlt0.2, n-14) or 112 (1.58*0.2, n=18), and higher
than in hiopsies from "norma1s"lPBC (1,6550.3). (p<O.OOOl).
NEM-insensitive PAP activities were not different in biopsies with
different degrees of statosis. However. NEM-insensitive PAP was
higher in cirrhotic hiopsies (5.83f0.4 unitslmg, n=19) than noncirrhotics (2.20t.0.!2. n=22) or "normals" (2.17f0.53. n=6);
(p<O.OOOl). In rdt liver. NEM-insensitive PAP activity was
unchanged in the 10 days following acute CC14 administration
(characterised hy intense liver regeneration and no excessive fibrosis). hut increased progressively following common hile duct ligation (CBDL). where lihrosis is the predominant feature (eg. day 28,
CBDL - 6.91 f 1.24. "Sham' - 1.97f0.3. pCO.oooO1).
Differences in activity of NIM-sensitive PAP conmhute to predisposition to AFL.. NEM-insensitive PAP activity may he involved
in transduction of the tihrogcnic signal following liver injury.
ZO 8-ADRENOCEITOR COUPLING IN THE IIUMAN
CARDIOVASCULAR SYSTEM
A FERRO (introduced by MJ BROWN)
Clinical Pharmacology Unit. University of Cnmhridgc. Addenhrmke's Hospibl.
Cambridge CB2 2QQ. England
P-Adrenocepbr (AR) antagonisls are widely usul in clinical practice in the
treatmutt of irhacmic heart disease ~d hypertension. the m o commonly
~
usal
being lhc @ I A R - r l s l i v eblockers. for example nlenolol or bimprolol.
(i) A prospective randomid study was performed in 6 healthy subjsts.
examining BAR subtype ~ponsesfollowing 14 days treatment with either lOmg
bisopmld daily M pl.Crb0. using a double-hlind cross-over dwign. 3 days after
cessation of treatmnt. subjbcts underwent trepdmill exercise ( h u r t rate response
being OIAR-medialal) and intravenous salbutamol infusion ( h u r t rate response
being @2AR-msdiatd). Exercise tachycardia was not different after bisopmlol or
placebo. whereas salbutamol-induced k h y c d i a was significantly greater after
bisopmlol. These results demonstrate Uut cardinc 02AR cross-sensitisation can
be induced prospectively. by prolonged 8 1AR hlockade.
(ii) PAR-mediated vasodilation was musured in human intemnl mammary artery
( M A ) and saphenous vein (SV). in v i m . using an organ-bath amngement. Both
IMA and SV were found to undergo predominantly B 2 A R - d i s t e d vuwdilation.
A novel finding WLI Ihal IMA could also undergo 01AR-mulisted vauxlilation.
as may SV to a I s u r extent. There were no differences in 4 l A R or in CZARmediated vasodilation hclwcen vsscls from DIAR-hlcrkal und nnn-BAR-hlockad
patients. Thus, P2AR cnms-unsitisation appears to he tissue-specific. nccurring
in myocardium but no( in these h l d vcsuls.
(iii) O A R - d i a l e d vasodilation was mepcured in human coronsry artery. in
vifm.using M organ-hath system. Bnth BIAR- and BZAR-mcdiatd vrwdilaticm
were Cmonslmted. Ihe PlAR component k i n g predominunt. Coronary arten*
were incuhated for 16 hours with 300nM CGP 20712A ( 8 specific BlAR
antagonist) and/or IpM noradrenaline (at this cimcentrdtinn. noradrenaline
activates almost exclusively P IAR). and Bl AR and U2AR-mlialal vauxlilatinn
were a
d suhxqucntly. B2AR respnns*s were unaltered following CGP
20712A: however. they w e n significantly desensiti.4 it)llowrng norddrenaline.
and this dcunsitisatinn was prevented hy cwincuhntinn with CGP 20712A. T h i s
B2AR descnsitisation o c c u d with no change in BlAR rapnnses. Thus.
sustained 0 1 AR agonism may cuusc crossJescnsitiwtion of 42AR respoehcs.
(iv) The n h u n h c e of stimulstory G-protein (Gs) nnd of inhihitory G-protein
(Gi) a suhunit isnfnnns. ~d nIw 01 G-protein 4 buhunits. were determined hy
SDS-polyacrylamide gel elstrophorcsis MJ immunohlntting of extrncts irom
human right atrial appendages. The resulting hnnds were u n n l y d hy l a w
densitomdry. No quuntitative diifcrenceb were Iound in GLor GI a or 4 whunits
in tissue from PIAR-hlcrkd I.riimparul with niin-UAR-hl<xkalpatients.
21 l l m m E m N I s l l M u A T E s ~
pRMoNocrrwsloNlN~HEART
AAGRAE
DepartnwtsdBiocfreniqadNLnficine,M&tydhm
A Na+-HCq-ceinflux carrierand the Na+-H+antiport have both
been shown to contribute to recovery from intracellularacidosis in
cardiac tissue. In this study the effects ofangiotensin I1 (10-1210-6M)
on proton fluxes and cardiac contraction have been investigated in the
isovolumic Langendd-perfused ferret heari both following
experimental displacement of intracellular pH (pHi) and during
reperfusion after myocardial ischaemia pHi was estimated using 3lP
nuclear magnetic resonance spectroscopy from the chemical shift or
intracellular deoxyglucosed-phosphate or inorganic phosphate. Net
proton efflux mtes were calculated at pHi 6.85 or 6.80. The
contributions made by different pHi regulalors have been examined by
comparing rates of pHi recovery in HCO3-- and nominally HCq--frcc
buffer systems and by using the high allinity Na+-H+antiport
inhibitor S(N-ethyl-N-isopropyl)amiloride. There was n o effect on
steady-state pH, following addition of angiotensin 11. Howevcr
angiotensin I1 caused a concentration - dependent slimulation
(maximum at
M: 67%) ofthe Na+-H+ antiport in nominally
HCQ--frce solution. Half-maximal stimulation of the Na+-H+
anti port occurred a1 lo9 M which is close 10 the &of thc cardiac
AT I receptor. Stimulation via this receptor was confirmed using
specific non-peptide blockers of the AT1 rcccptor. Angiotensin I1 also
stimulated pH, recovery rrom an acid load whcn HC03'-dcpcndcn1
p H i regulators wen: functional. Howcvcr in both SCL$ o f pcrrusion
conditions (HCO3-and HC03--free) rccovery of'contrxticmwas
dclaycd by the addition of high concentrations of angiotcnsin I t .
During reperfusion following myocardial ischacmia. angiotcnsin I 1
significantly increased proion extrusion but impaired contrxlilc
rccovcry. The rcsults show that angiotensin II faciliriltcs proton
extrusion in the heart. Although this may haw bencficial c f f c c ~i n
maintaining homeostasis under physiological conditions the astxiatcd
NJ+-influn could cxaccrbate cellular dysfunction and impair thc
rccovcry of contnction following mytrardial ischacmia.
-
22
CAN
NITRIC
OXIDE
MODIFY
PATHOPHYSIOLOGY OF VASCULAR INJURY?
I'H GROVES
THE
(Introduced by AH HENDERSON)
University of Wales College of Medicine, Heath Pwk,
Cardiff, CF4 4XN
Balloon angioplasty causes plaque rupture, medial
dissection and local platelet-thrombus formation. The
reparative response involves the proliferation and
migration of smooth muscle cells (SMC) which, if
excessive in the clinical context, may result in restenosis.
Adherent platelets are a n important source of growth
factors which stimulate vascular SMC. Nitric oxide (NO)
inhibits platelet adhesion and also SMC proliferation i n
vitro. The effects of exogenous NO on the vascular
response to angioplasty were therefore investigated. The
NO donor SIN-I was shown to increase platelet cyclic GMP
(p<0.005) and reduce platelet adhesion (p<0.005) and
platelet-thrombus formation (pc0.05) at the site of pig
carotid angioplasty. The time course of subsequent intimal
thickening and SMC proliferation was then characterized
and both were shown to be closely related to internai
elastic lamina (IEL) rupture. Molsidomine (whose active
metabolite is SIN-1) was shown to increase arterial wall
cyclic GMP (p<0.05). When the !EL remained intact