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M30 INTRACEUULAR AMMONIUM PRODUCES TEE
DEPoLARISA~ON
AND CONDUCTANCE CHANGES
OBSERVED IN XENOPUS LAEVIS oo<TyTES EXPOSED TO
EXTRACELLULAR AMMONIUM
S BHANDARl and M HUNTER
9P
ROMKI. Struchrral diffcrmces bctwscn ROMKl and ROMK2 lie in the
N-tuminus, with ROMKl containmg an additional 19 amino acids. This
suggests that the N-terminal region may intaact with the selectivity filter
of the pon to &kct k u m blockage or this q i o n is dbxtly involved
in the k u m - b i o d i n p site.
Dr Sunil Bhandari is in receipl of a NallOM/ Kidney Research Clinical
Training Fellowship.
Department of Physiology, University of Leeds, Worsley Medical and Dental
Building, Leeds,LS2 9NQ, UK
Oocytes posscss endogenous transport system but the mechanism responsible for
ammonium induced de-polarization and conducmcc incrrare remains m a n s w e d
Na'K'CT cc-transport in place of K' and movement via K' channds have been
proposed. We examined the e&t of ammonium (NH*+)in uninjected Xenopus
cacytcs to elucidate the mecbanism producing this dtpolarizationand conductance
Both hvo-dectrode voltage ckmp and maaopstch techniques were utilized. Stage
V M oocyta were defoUiculued in coUa&cnasc lmglml ( S i p type IA).
Exnrcclhrlar W' as ammonium hydroxiddchloride 3OmM waa applied in Ringers
solution (in mM NaCl 97, KCI 3, M&l2 2, CaCI, I, HEPES 10) and in
scdium/ehloride frre solutions (in mM: M&12 2, CaCh I. HEPES, 10 KCI 3, NMDG
10, gluconic acid lactone 10). S o l ~ twere
i ~ all
~ titrated to pH 7.4
NMDG and
gluconic add k o n e . A range of transport and channel blockers were applied and the
pH dfea,for a pH range of 6.4-8.8, was sxamincd. Cdl msched and excised inside
out mncropatches were examined using the above solutions. Pipette solution
consisted of in mM: KCI 100, MgCIz 2, CaClz I. HEPES 10
Application of W' produced a concentration dependent effect with a calculated kd
of 27mmovI and &y of 17pS (n=5) using the Maechadis Menton Equation and
curve fitting plot. 3OmM W' led to a fall in E, ImM Ba", 50pM strophamhidin
and 1pM Mliloride produced no &en. 50pM frwemide eaused a 16% reversibly fall
in slope conductance, while ZmM DPC produced a significsnt fall in slope
conductancefrom 5.3M9pS to 2.5M.75pS and E,changefrom -4OmV to -7OmV.
In macropatch analysis, addition of e x t d l u l a r W' to cell attached patches
d slope conductance from 046M.lpS to1.48M.3pS (d).
produced M i
Excised inside out patches also produced a similar incmased slope condudance
shifted to more nc@tive potentials on
(n=7); 0.49-H.ZpS to 1.29fo.5pS
application of either ringers or N&* This effect was reversibly inhibited by 0.5 mM
DPC (n%).
Depoluization and conductance changes observed in whole cell studies of Oocyte
with extracellular NH,' are due to secondary ch.ngcs in innacellular W'.
Ammonium entry in part via cc-transporters and from non-ionic ditfusion may be
responsible for the observed secondary &ect
Can should be taken whm assessing the permeability of TW' through c h m l s or
transporterswhen e x p d in Xenqpus Oocytes.
Dr. Sunil Bhondmi is in receipr of a Natioml K i h y Research Clinical Training
Fellowship.
+
M3 I Involvement of the N-terminal region in ROMK
potassium channels to caaium sensitivity.
S Bhandari and M Hunter
Department of Renal Physiology,
M32 THE RELATIVE IMPORTANCE OF NITRIC OXIDE
AND PROSTAGLANDIN ACTIVITY IN DETERMINING
ENDOTHELIAL FUNCTION IN HEALTHY ADULTS
'DR
MEEKmG,
'S
ALLARD,
'J
MUNDAY,
CHOWIENCZYK, 'KM SHAW AND *'MH CUMMINGS
2P
'Departments of Diabetes and Endocrinology, Queen Alexandra
Hospital, Portsmouth, and 'Clinical Pharmacology, UMDS, St
Thomas' Hospital, London ( * = introduced by)
The vawdilalon. response to acetylcholine IAChl is widely used 10 investigaie
the L-Arginine-nitric oxide pathway bul is inwmplctely inhibited by nitric
oside synthase inhibitors. suggesting that other niedialon are involved. We
esainined tlie role of prostaglandms in the vasodilaton. responsc 10 ACh Seven
healthy nondatetic. noriiiotensive adults were studied (2M 5F. age 30.4f 5.7
yr linean f SDI. total cholesterol 4 5 f (1.6 mnioVI. triglyceride 1.2 f 0.4
minolll). Forearm blood flow lFBFl was measured using a venous occlusion
plcthysinograph! iechnique. Vasodilalor responses were iiicasured at baseline
and following lmal intra-anerial (brachial artery) infusions of ACh (7.5. 15. 30
incgliiiin) Subjects' vasodilator responses at baseline and after ACli were then
reitsanlined following local inlra-arterial infusion of indoniethacin IINDJ
(~1.3niglI(lOtnlforeami volume over 20 niin). a n.clo-o.\-\.genaseinhibitor.
Students paired I test was used to winpare baseline and ACli tsaasodilator
responses before and aner IND infusion. Absolute values of FBF wcrc
esainined. Following IND. the vasodilator responses to all ACh doses were
signilicantl? reduced.
In conclusion. forearm vasodilatory
sublects are attenuated following IND
responses to ACh in bealth! control
infusion. This suggcsis thal vascdilaton.
proslanoids are involved in tlie endothelial response to ACh
University o f Leeds,L e d , UK.
ROMKI and ROMK2, lowconductance inwardly rectifying K' channels
(IRK) cloned from the rat kidney, regulate recycling and d o n of K'
ions across the apical membrane of the thick escmding limb of the loop
of Hcnle and d i d nepbron. Previously we have demonstrateddiff~ences
in selectively to thallium in ROMKl and ROMK2. In the present study
the ionic flux of caesium, potassium chanml blocker, was examined.
5Onl of ROMKI and ROMK2 cRNA were injected into collagenase
(Img/ml) treated Xenopus oocytm. Investigation using two-elcctrodc
voltage clamp (TEVC) confirmed expression of barium-scnsitive K'
cwmts & 48 hours of incubation at 19OC in mcdified Bath's
medium. Bath solution contained in mM: 1OOKCI. IMgCIZ, ICaCI2,
IOHEPES tihated to pH 7.4. Caesium in a ummtration range I-1OOmM
was added to the bath solution for blocking studies. Results are given as
mcaaBEM.
Caesium produced concenmtion and voltage d-dent
block of inward
aurenb in both ROMKl and ROMK2. The Hille coefficients ranged
from 1.2fo.2 at -12OmV to 1.12M.1 at -6OmV for ROMKl and
0.84fo.03to 0.77fo.03 for ROMK2 rcspeCtively. These values for each
channel were. not significantly different from each other w . 7 and 0.1
respeaively). The Kd (concen-m
of half-maximal block) decreased
with hypapolsrisation in an exponential fashion. Dose response curves,
fitted using the Hillc equation, gave mean Kd values at OmV of
100.6f16.6 ( n 4 ) and 30.li5.7 ( n 4 ) for ROMKI and R O W
respeaively (pcO.05) and elecbic distances (6) of 0.94fo.06 and
1.27fo.05 respdvely. From the Woodhull equation for voltage
dependent block the values of 6 ranged from 0.87 to 0.94 and 0.67 to
0.98 for ROMKl and ROMK2 rcspeCtively for the concentration mange 5
to 50 mmoVI.
ROMK2 is significantly more sensitive (lower Kd) to caesium than
M33 PERIOPERATIVE CARDIAC RISK
STRATIFICATION, WPERCOAGUABILITY AND
HEART RATE VARIABILITY
N MAMODE, G DOCHERTY, P W MACFARLANE, G D O
LOWE AND SM COBBE
Peripheral Vascular Unit, Glasgow Royal Infirmary, Alexandra
Parade, Glasgow G31 2ER, Scotland
Current methods of preoperative cardiac risk assessment are inadequate;
radionuclide scanning remains the gold standard. The aim of this study
was to determine the value of clinical risk factors, levels of haemostatic
markers, heart rate variability and thallium scanning in the prediction of
perioperative cardiac risk.
297 patients undergoing surgery for peripheral vascular disease were
prospectively entered into the study and screened for perioperative
myocardial infarction using daily cardiac isoenzymes (CK-MB) and
electrocardiograms for the first 3 days after surgery. Outcome measures
were proven myocardial infarction or cardiac death within 30 days of
surgery. Secondary outcome measures were len ventricular failure,
unstable angina or life-threatening arrhythnuas within 30 days of surgery
Preoperative data collection included clinical risk factors, levels of
fibrinogen, fibrin degradation products(FDPs), von Willebrand Factor
and plasma viscosity. Patients also underwent preoperative ambulatory
electrocardiography and radionuclide scanning.
2 I patients sustained a perioperative myocardial infarction or cardiac
death, and a further 20 patients had a secondary penoperative event. A
logistic regression model identified the following as independent