Download Vertebral Osteoporosis: Factors Affecting Urinary

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studies mggest that additional genetic factors are actiog to produce the disease
phenotype. We have show that the HLA alleles and CD do not cc-segregate in
families multipb al€&
with CD.Suggesting that the HLA association is entirely due
to the necessity to have these DQ alleles in orda to eapress CD, but that the main
genet;c&kpcsitia~k atalmsotherthao theMHC. We hypouKsisedthat some
of the insulin depeoacnt diabeta mllitus (lDDh4) susceptibility loci d d be
common to CD since CD is fwod in loo/. of IDDM patienls. In order to test this
hypourdslwenty-fiveEuropean two- or three-generation,multiply affectedfamilies
were shdied using fluoresccntly labelled microsatellite markas linked to theloci
Mplicptedin D D M These miuosatellitawere analysed using an ABI fluorrscent
DNAsquartr. subdinicslCD was identified in the familiesusing anti-endomysial
g with Qodcnalbiopsy of positive cases HLA typing of the DQ
antibody d
lod was performedby PCR and stquenocspeciIic oligonucleotide hybridization In
tbc~~8nalysisDQ2acgafivesubjectswactrratalashavingunlmown~ection
status. This p m t s any of carriers of the CD gene who do not have the HLA
swxpliiilityallelesiiun beinga-nmled as ncombinaots thus redXingthepossibility
ofafalseacgativedt Ldsooreanalysiswapperfdusingh4LINK,assumiog
dominanttrananisSmwithWhpeaehaaceaadagene~ofO.O1.
ModeliitewalysiPwPspwing~wiichrepwtsa~1-6eelodsmeand
themaximumkd soac obtained f a any h.aasmissioamodel. We have examined the
~D6S264.D6S273.D6S290.D6S291.D8S88.D8S257.D8S556.D10S197.
DlOSUo.D1 lS922,D13S122. D13S158. D I W , ESR and TH, which have been
implicated m the genet;c aetiology of D D M by Davis et al(1994). and have found no
evidence to mggcst that any of these loci are linkedwith susceptibility to CD.
M58 EFFECT OF INSULIN ON THE PZ-ADRENOCEPTORMEDIATEDCAMPRESPONSE INBC3HI CELLS
HE HOPKINSONand SJ HILL
17~
or supranormal urinary outputs o f calcium, although the mean
plasma 1.25(OH)zD level of the subnormal group is lower than
in the others combined (Hine TJ et al Clin Sci 74 Suppl IS, 741,
19SS). Patients in the latter two groups (5 male, 15 female; age
23-83 yr; 6 supranormal) have therefore been studied by six-day
balances. Calcium intake (1 1.9-59.4 inmollday) was that
normally ingested, as assessed by a metabolic dietitian. Patients
were not taking vitamin D, fluoride, corticoids or diuretics.
Nine were in neutral balance(tlO% intake) 7 positive and 4
negative.
Urinary calcium output (2,s-9.55 mmol/day) was significantly
negatively correlated with plasma 1,25(OH)2D (41-197 pmol/L)
in the combined groups, r = -0.494. p<0.05; in a subnormal
group the reverse is known to occur (Hine TJ et al Clin Sci 86
Suppl 30, 24p 1994; r = + 0.607: pcO.001). Urinary calcium
was.positively correlated r = +0.723. p<O.Ol. with plasma 25OHD ( 1 5 4 7 nmol/L) in the normal group. negatively with the
plasma 1.25(OH)zD/PTH ratio (0.74-4.03) in the combined
groups (I=
-0.566. pc0.02) and positively (r = +0.6lS.p<0.02)
with net calcium absorption (9.7-42.7% of intake) in the normal
group. Urinary calcium was not significantly correlated with
calcium intake ( r = +0.237), urinary Na' (r = +O. 176). age
(r = -0.3S7). plasma PTH (r = + 0.409) or balance
(r = - 0.274) in the combined (or either) groups.
Two interesting, unexpected points emerge: plasma 25-OHD
has a role in urinary calcium excretion in normocalciuric
osteoporosis, and the role of plasma 1,2S(OH)zD in calcium
excretion differs in patients with differing outputs o f calcium.
Department o f Physiology and Pharmacology, University of
Nottingham, NottinghamNG7 2UH, England
Previous work using fractionated cell membrane preparations Gom transfected cell
lines has suggested chat phosphorylation of the Pradmoceptor by the activated
insulin receptor yosine k m c leads to an attenuation of isoprenaliistimulated
daylyl cyclase activity (Karoor V et al, JBC 270(43):25305-25308). The present
shdy employodh DC3Hl ccll lie which is established as a smooth muscle model
and express awrogaxxlrbbotha hmaiaralinsulin receptor and a P,-adrenoceptor.
In this communication we show that insulin produces an augmentation of the
maximum isoprenalie-stimulated accumulation of cAhW in intact BC3Hl cells.
M J H l cells were grown to confhnxr in 24 well plates and loaded with 'H-denim.
Cells wue stimulatedwith isoprenaline for 1O'minuteswith or without pre-exposure
to lOOnM soluble insulin (porcine Velosulm) for 30 minutes. Tritiated CAMPwas
isohled tkm whole cell lysaks by sequential Dowex-Aluminn chromatography. The
presence of functional insulin receptors was confmed using a3,H-2-deoxygluuKe
w studies wcre
uptake assay, and competitive whole cell insulin receptor b
perfmed using 'BI-Iabelled porcine insulin.
The EC, value for isoprenaline was 70 f Inh4 with maximal CM
accumulation
occmingat a concentration of IOpM. The specific ~a-amenoeeptOr
antagonist ICI
118,551 caused potent inhibition of isopnnaline-stimulatedcAhW produaion (KD
= 0.lnM). D a d 1 H - 2 d ~ x y guptake
l ~ ~by
~ h e =HI cells was m
d by
45% i 6% @< 0.01) in response to IOOnM insulin M a m a t of l'I-ins~li
b
eamlimedthe presena:ofbigh affmity binding sites (IC, 30 f 0.5pM). Pre
m d o n o f B C 3 H l cellswith lOOnMinsulincauseda263%increasc(pc0.01)
in the maximum isopdine-stimulated accumulation of cAhW, but there was no
eKect of insulinon the EC,or slope of the isoprenalineQse rrspoase curve.
This stuay demonsbates a small but significant potentiation of the isoprenali
stimulated pa-admmccptor response by insdin, in intact cells with ecdogcnourly
scpressedreceptors. These &conW i h prevlws reports in trwsfected cell
membrane preparations, but are in keeping with data from pemcabilized human
lymphoqtes(FeldmanRD,BJP 110:164M644 1993). Cross-talkbdweentyrosine
kreceptors and 0 proteibcoupled receptors is k c r e a s i d reoognisedas an
important p b m e n o n , and insulin-mediated augmentation of the p2-admloccptm
responrc in vivo could be implicated in the Link between insulin resistaace and
essential hypertension.
M59 VERTEBRAL OSTEOPOROSIS: FACTORS
AFFECTINGW A R Y CALCIUM EXCRETION
T J Hine (introduced), N B Roberts and W H Taylor
Departmentso f Clinical Chemistry and Medical Microbiology.
Royal Liverpool University Hospital. Liverpool L7 S X P .
I t is unclear why, at
presentation, osteoporok patients exhibit
subnormal, nornial (2.5-6.25 minollday. female; 3.0-7.5, male)
M60
TWO NEUROPEPTIDE Y (NPY) ANTISENSE
PHOSPHODIESTER
OLIGODEOXYNUCLEOTIDES
ARE
INEFFECTIVE IN INHIBITING NPY GENE EXPRESSION IN
THE RAT HYPOTHALAMUS
Simon Dryden, Lucy Pickavance, David Tidd', Gareth Williams
Diabetes & Endocrinology Research Unit, Department of
Medicine and 'School of Biological Sciences (Biochemistry),
University of Liverpool, UK.
Neuropeptide Y (NPY) is a potent appetite stimulant when
injected into the hypothalamus, especially the paraventricular
nucleus (PVN). The arcuate nucleus (ARC) is the main site of
NPY synthesis, and there are NPY neurones projecting
upwards to the PVN, the main site of NPY release.
Antisense oligonucleotides appear to inhibit gene expression
in vitm, and in some cases, in vivo. This study aimed to
compare the effect of an NPY-specific antisense unmodified
phosphodiester (D-ODN) and a similar antisense propylprotected phosphodiester (P-ODN) on food intake body weight
and NPY gene expression.
For both ODNs, adult male rats (n=8/group) had the antisense
ODN (24 pg/day) infused via osmotic minipumps for 7 days into
the 3rd ventricle. Control rats (n=8/group) had either saline or a
missense ODN (24 pg/day) infused under similar conditions.
The rats were sacrificed after 7 days and NPY mRNA levels
measured by Northern blotting.
There were no significant changes in food intake, body weight
or NPY mRNA levels in the antisense D-ODN-treated group
compared with either of the control groups.
Food intake and body weight were reduced by 9% and 6%
respectively (p<0.05) in the antisense P-ODN-treated group
compared with the saline-treated controls. NPY mRNA levels
were unchanged in both the antisense and missense groups
compared with the controls.
These results suggest that these phosphodiester ODNs may
not be effective in influencing their target mRNA, and therefore
exerting a specific effect on hypothalamic NPY in the rat.