Synthetic tumor-derived human hypercalcemic factor exhibits parathyroid hormone-like vasorelaxation in renal arteries

Vol. 149, No. 1, 1987 November 30, 1987

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages

227-232

SYUTHRTICTUWOR-DERIVED HDHARHYPERCALCEWIC FACTORRXHIBITS PARATHYROID HORMORE-LIKE VASORBLAXATIORIN RBRALARTERIES RaymondJ. Winquistl*. Elizabeth P. Baskinl and George P. Vlasuk2 Departments of Pharmacology1 and Biochemistry2 Merck Sharp & DohmeResearch Laboratories West Point. PA 19486 Received October

15,

1987

sumARY: Synthetic human tumor hypercalcemic factor (l-34, hHF) was compared with parathyroid hormone (human sequence, l-34; hPTH) for vasorelaxant activity in isolated rabbit renal artery segments. The hHF exhibited a potent = 1.3~10-~Hl and profound (98%) relaxation which was significantly (IC50 greater in magnitude than that obtained for hPTH (IC5D = 4.5x10-9H; maximal relaxation = 78%). The relaxations to both peptides were concentration-dependent and not associated with changes in cyclic AMP levels. These results demonstrate a parathyroid hormone-like response, independent of adenylate cyclase activation, in isolated renal arteries. Renal vasodilation may be important for the effects on renal function shared by these two peptides. o 1987 Academic Press, Inc. The hypercalcemia

associated

caused by a tumor-derived similar

from these patients

terminal

cell

acid

significant

lines

factor

disease that

hormone (PTH).

tumors (1) as well as a protein

bone-derived

malignant

circulating

to those of parathyroid

2) derived

with

sequence analysis

of

the

isolated

radioimmunoassays (3) implying other structural factor

successfully

isolated

and PTH.

Recently,

and characterized

predicted

to

encode the

full

expression

of

the corresponding

to be actions

from excised

tumor cell

line (BEN;

adenylate cyclase in

to PTH. Although partial

homology with PTH (21, the tumor factor

circulating

Both extracts

in stimulating

which are responsive

believed

has biological

released from a cultured are active

is

peptide

factor

amino

disclosed

cannot be detected by PTH

distinctions

between the

using BEN cells,

Suva et

al

(4)

a complementary DNAclone which was

length

PTH-like

tumor

protein

in a mammalian cell

Abbreviations: hHP (synthetic human hypercalcemic (synthetic humanparathyroid hormone (1-341). *To whomcorrespondence should be addressed.

factor.

factor

Following line (l-34));

PTH-like hPTH

0006-291X/87$1.50 221

Cowripht 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 149, No. 1, 1987 activity

was

documented

osteoblast-like Suva this

et

al

(4)

response most

on

line

will

allow factor

for

isolated

renal

artery

assays

for

the

PTH,

which

is

concert

calcium

absorption.

synthetic

hHF

segments.

In

associated

with

of cyclic

AMP in

with

The disclosure

AMP of

of

PTH exerts

a potent

segments

known

the

with

known

the

formation

the primary

physiological

in

an

sequence

by

functions

since

activation

of arteries

tubular

the

exposed MBRIALS

in

effects

using

the

of

to be among

laboratory of

elicited (7),

to these

peptides.

the

Renal

animals

(8).

PTH on phosphate

vasorelaxant

cyclase

vasorelaxant

of PTH (6-8).

isolated

vasodilation

adenylate

appears

actions

compared

(l-34)

and profound

which

prominent

we

hPTH

(5)

vasodilator

typically

addition,

renal

cyclic

elucidation

Therefore,

(l-34)

of

RESEARCH COMMUNICATIONS

(hHF). that

to in

(UMR106).

found

vasodilation

AND BIOPHYSICAL

stimulation

recently

sensitive

act

by

cell

hypercalcemia We have

may

BIOCHEMICAL

and

effects

renal by

we measured

of

artery PTH tissue

ring may

be

levels

AND METHODS

Male New Zealand White rabbits (2.2 to 2.6 Kg) were killed by asphyxiation and the renal arteries were rapidly removed and placed in physiological salt solution (PSS) of the following composition (in mM): NaCl (130); KC1 (4.7); KH2P04 (1.18); MgS04e 7H20 NaHC03 (14.9); (1.17); CaC12 (1.6); dextrose (11.0); Na2EDTA (0.026). The pH was corrected to 7.3 with 1N NaOH and the PSS was continually gassed with 95% O2 and 5% CO2. Renal arteries were cleaned of extraneous tissue taking care not to perturb the luminal surf ace. 41nm ring segments were carefully mounted on fine (0.0085) stainless steel wires which were held in Plexiglas holders suspended in jacketed organ baths (50 ml) kept at 38OC. Renal artery rings were connected via surgical silk (4-O) to Grass force-displacement transducers (FT.03) for monitoring changes in isometric force. Rings were stretched (2.0 grams-force) to an optimal level of resting force as determined by maximal responsiveness to a test concentration of a contractile agent (methoxamine). Following a 7. hour equilibration period, renal artery rings were contracted to a 50% maximal response methoxamine which averaged (ECso) by approximately 1.5 grams--force. Tissues were then washed several times over a 30 minute period after which the same concentration of methoxamine was reapplied. A cumulative concentration-response experiment with either hHF or hPTH was begun once the contractile response to methoxamine reached an equilibrated plateau. Each renal artery was used for only one dose-response experiment. Concentrations of peptides eliciting 50% relaxation of the methoxamine contraction (i.e. IC50) were obtained by linear regression using log transformation of the concentrations. Statistical evaluation of the data was performed by the Student’s t test for unpaired samples using geometric means of the IC50 values and the 0.05 level of probability as the level of significance (P < 0.05). A synthetic sequence encompassing residues l-34 of the amino-terminus of hHF (4) was used and is described by Horiuchi N. et al (9). hPTH (l-34) was purchased from Bachem. The purity of each peptide as well as the documentation of stock solution concentrations was accomplished by amino acid analysis. 228

Vol.

149.

No.

1, 1987

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Measurements of cyclic AMP were made in ring segments exposed to varying concentrations of hHE', hPTH or forskolin for 5 minutes. Isobutylmethylxanthine (LBMX, 10h6M) was included in the PSS during these experiments. Tissues were immediately frozen in liquid nitrogen and homogenized in 1 ml of cold 6% trichloroacetic acid. Homogenates were centrifuged and the supernatants extracted with water-saturated ether and lyophilized overnight. The dried samples were a-* -*ayed for cyclic AMP using a non-acetylated RJA (New England Nuclear). TCA insoluble total protein was measured by the method of Lowry et al (10) following solubilization in 0.4N NaOH. Methoxamine was supplied as a gift from Burroughs Welcome (Research Triangle Park, NC). IBMX was purchased from Sigma Chemical Company (St. Louis, MO), and forskolin from Calbiochem (San Diego, CA). RKSULTS __--_.Both

hHP

contractile (Fig.

to

The

1C5o

less

that

than

methoxamine the

contractile relaxation

caused

concentrations for

force

isolated

(L.SXLO-~M)

hPTH

relaxation

experiments

for

with

not (hHF

was

(~.~xLO-~MI.

obtained

did

(i.e.

differ :

hPTH

lo-'M

to

(9821%)

between

artery

the

than

but

hPTH at

Fig.

not relaxation

was

significantly

The

levels

of

two

groups

prior

hPTl1

1).

of

O-O 0-O

1

6

5 g cr E

=

to

4020 1

-10.0

-8.0

-9.0

-7.0

LOG [PEPTIDEIM

Figure 1. The relaxation response to hHF (n=4) and hPTH (n=7) in isolated rabbit renal artery rings contracted by methoxamine. The ordinate is expressed in terms of precent maximal relaxation of the methoxamine contraction while the abscissa is log concentration of the particular peptide in molarity. Values are means + standard error of the means. An asterisk designates a significantly greater relaxation to hHF compared to hPTH (P< 0.05). 229

the

the

1.51+0.0s

hHF hPTH

0I

The

methoxamine

z 805w n: 60z 2

the

greater

grams-force). 100

the

significantly

maximal

grams-force;

of rings

~xLO-~M,

slightly

(78+5%).

L.46_+0.07

renal

relaxations

The hHP

decreases

rabbit

larger

tested

hHY

for

in

significantly

contraction maximal

concentration-dependent

to methoxamine

hHF elicited

high

calculated

than

hPTH

response

1).

middle

and

Vol. 149, No. 1, 1987

BIOCHEMICAL

AND 8lOPHYSlCAL

RESEARCH COMMUNICATIONS

TABLEI hHF

hPTH(l-34)

cAHP (fmole/ma) 10226 k 1108

Control IPeptid~Jh 1x10 -8 1x10 -7 1x10

c&P

Forskolin

c~(fmolelms1

(fmole/mg)

10226k1108

7538 k 916

6726 f. 211

5713 + a49

6652 + 247

6239 + 461

7007 + 226

10226+1108

Forskolin*

113142 fr 792**

Cyclic AHP levels were measured by radioimmunoassay as described in Methods. Protein was measured by the method of Lowry et al (10). *Final concentration of forskolin was 10%. Numbers represent the mean of 3 independent determinations + s.E.H. **p < 0.05.

Incubation

of renal artery

ring

segments with several

to 10V7W) of both hPTH and hHF failed of cyclic

AMP (Table 1).

adenylate

cyclase

significant

Forskolin.

was added as

concentrations

to significantly (lo-%I)

tissue

which is a direct

a positive

increase in the levels of cyclic

alter

control

AMP (P(

(up

levels

activator

and caused a

of large

0.05).

DISCUSSIOU The biochemical

similarities

(IUD41 and hyperparathyroidism circulates

(e.g.

and is responsible

the recent

isolation,

residing

with PTH antisera

appears sufficient to adenylate

and molecular

in the carboxy (e.g.

3).

factor

This was borne out with

cloning

(2.4)

terminal

of

the MF

possibly

portion

due to

of hHF does

However, the amino terminal homology with the PTH receptor

in bone-derived

of malignancy

a PTH-like

homology with hPTH but,

for hUP to interact

cyclase

suggested that

HHH syndrome.

the

amino terminal

differences

not interact

for

1.3)

purification

which has significant structural

between humoral hypercalcemia

cell

lines

which is coupled

and renal cortical

membranes

(4,9). We have now shown that isolated

renal

several

concentrations

arteries hPTH.

arteries

(Pig.

hHP also exhibits 1).

It

is of interest

activity

The hHF was more effective

and produced almost total

which was a significantly

PTH-like

larger

to note that

relaxation

in relaxing than hPTH at

of the contracted

response than that

obtained with

hHP is 6-10 times more effective

230

than

Vol. 149, No. 1, 1987 PTH

in

producing

reason

for

clear

BIOCHEMICAL

this

since

enhanced

neither

Therefore,

for

at

shown)

the

to hHF in

caused

in

the

an approximate

of

lo-‘M

this

arteries

cyclic

is in

is

in

increase

in

the

not

at present

appear

not

to

of

cell

line

to be

forskolin

relaxation

level

The

AMP levels.

contrast

an 80-10096

(9).

cyclic

nucleotide

This

elicited

10 fold

rats

changes

relaxation.

of

renal

significant

level

observed

RESEARCH COMMUNICATIONS

thyroparathyroidectomized

response

a concentration

and

in

peptide

differences

responsible which

hypercalcemia

AND BIOPHYSICAL

(data

cyclic

not

AMP (Table

I). As with that

the

hHF is

receptor shown

the

in

results eliciting

assays

of antagonist display

analogs

ion

of

not

in failure

renal

may

indicate

mechanism

AMP in been

vascular

reported

regional

tissue

utilized

endothelium

for

Our renal

results

the

effects

phosphate

the

of

demonstrate

threshold

and all

particular

et

elevate

vascular

vascular

spasmogen

(e.g.

require

the

presence

these

peptides alter

AMP levels receptor

which

have

by

renal

methoxamine, presence

cyclic

may indicate

induced in

in

and/or

cells

hHE’,

the

unpublished

muscle

relaxation to

the

in

investigators

been

and &

increases

smooth

ostensibly

to

vitro

al.,

PTH

has

to

cyclic of

PTH

this

arteries serotonin.

of

an

intact

(5). a functional,

vasodilation

peptides or

of

fail

PTH-induced

(11)

and

if

to hHF as well

response these

PTH,

since

and

subclass

tissue.

mechanism

(12))

(Winquist, hHE’ to

of in

feel

the

study

to hHF in

amide

we

with

this

3 variety

activity

or

not

in

relaxation

different

appears

efficacy

Renal

in

the

and

full

arteries.

filtration

but

to

of

histamine)

hPTH

particular

relaxation

independent

the

preparations,

in

in

arteries

3

by some (6,7)

The

activity

assays,

interaction

hHF used

relaxation)

of

this

differences

peptide. occurs

in

a direct

PTH (7-34)

renal

The

arteries

via

examined

(i.e.

hPTH

derived

synthetic

PTH (e.g.

agonist to

bone

PTH- like

observations).

coupling

The

We have

partial

relaxat

tissue.

comparable

(9).

in

vasorelaxation

vascular

to possess

fi,vo

obtained

enhancing

on renal

PTH-like

response

associated

with

as to PTH may play function

phosphate 231

such excretion

of hHF in enhanced

glomerular

an important

as either (e.g.

role

lowering 3).

isolated

in

renal

Conversely,

BIOCHEMICAL

Vol. 149, No. 1, 1987

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

the renal vasodilation

may be a necessary response to both mitigate

vasoconstrictor

of the calcium load which is reabsorbed and thus ensure

delivery

effect

any renal

of the calcium to systemic sites. ACKNOWLEDGMENTS

We would like

to thank Drs. M. Caulfield,

Jay Levy for providing amino acid analysis,

the synthetic

hHF (l-34)

M. Rosenblatt, peptide,

R. Nutt and Mr.

Lori-Anne Wassel for

and Carole Hannan for her competent secretarial

support.

REFERENCES 1.

Rodan, S.B., Insogna, K.L., Vignery, AM-C., Stewart, A.F., Broadus, A.E,, D'Souza, S.M., Bertolini, D.R., Mundy, G.R., Rodan, G.A. (1983) J. Clin. Invest. ~,1511-1515. 2. Hoseley, J.M., Kubota, M., Diefenbach-Jagger, H., Wettenhall, R.E.H., Kemp, B.E., Suva, L.J., Rodda, C-P., Ebeling, P.R., Hudson, P.J., Zajac, J.D., Martin, T.J. (1987) Proc. Natl. Acad. Sci. USA 84.5048-5052. 3. Stewart, A.F., Horst, R., Deftos, L.J., Cadman, E.C., Lang, R., Broadus, A.E. (1980) N. Engl. J. Med. 303.1377-1383. 4. Suva, L.J., Winslow, G.A., Wettenhall, R.E.H., Hanunonds, R.G., Moseley, J.H., Diefenbach-Jagger, H., Rodda, C.P., Kemp, B.B., Rodriguez, H., Chen, E.Y., Hudson, P.J., Martin, T.J., Wood, W.I. (1987) Science 237.893-896. 5. Wallace, A., Vlasuk, G., Faison, E., Winquist. R. (1986) Pharmacologist 28,161 (Abstract). 6. Nickels. G.A. (1985) Eur. J. Pharmacol. 116.137-144. 7. Nickels, G.A.. Metz, M.A., Cline, W.H., Jr (1986) Endocrinol. 119.349-356. 8. Wang, H-H., Drugge, E.D., Yen, Y-C., Blumenthal, M.R., Pang, P.K.T. (1984) Eur. J. Pharmacol. 97.209-215. 9. Horiuchi, N., Caulfield, M.P., Fisher, J.E., Goldman, M.E., McKee, R.C., Reagan, J. E ., Levy, J.J., Nutt. R. F.. Rodan, S.B., Clemens, T.L. and Rosenblatt, M. Science in press. 10. Lowry, O.H., Rosebrough, N.J., Fart-, A.L., Randall, R.J. (1951) J. Biol. Chem. =,265-275. 11. Stanton, R.C., Plant, S.B., McCarron, D.A. (1985) Am. J. Physiol. E.E822-E826. 12. Goldring, S.R., Roelke, M.S.. Bringhurst. F.R. and Rosenblatt, M. (1985) Biochemistry 24.513-518.

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