Clinical Science (1981) 61,299s-301s
299s
Activation of renin in an anaplastic pulmonary
adenocarcinoma
F. S O U B R I E R ' , S . L. S K I N N E R ' , M. M I Y A Z A K I ' , J. G E N E S T 3 ,
J. M E N A R D ' A N D P. C O R V O L ~
'INSERM U 36, Paris, France, 'Department of Pharmacology, Shiga University of Medical Sciences, Japan, and 'Institut de
Recherches Cliniques de Montrkal, Montreal, Canada
Summary
1. Biochemical characteristics of a renin-like
enzyme secreted by a pulmonary adenocarcinoma have been studied. A very high renin
content was revealed by both enzymatic (10.4
Goldblatt units/g of tissue) and direct radioimmunoassay of immunoreactive renin (23
Goldblatt units/g of tissue).
2. The higher value by direct radioimmunoassay suggested the presence of an inactive form
of the enzyme. Indeed 40-100% activation
occurred with treatment by trypsin or pepsin or
prolonged dialysis at pH 7.4 with or without
prior acid dialysis. This neutral activation was
completely abolished by a serine proteinase
inhibitor.
3. A large fraction of the renin in this tumour
is inactive. In comparison with other prohormones produced in tumours the findings
support strongly the proposition that renin passes
through a proenzyme step in synthesis.
Key words: adenocarcinoma, renin, tumour.
Abbreviations: A N G I, angiotensin I; G.U.,
Goldblatt units; PMSF, phenylmethylsulphonylfluoride.
Introduction
The existence of an inactive form of renin has
long been recognized, having been described by
Lumbers [ l ] in human amniotic fluid and by
Skinner et al. [2] in plasma.
However, the presence of an inactive 'form of
renin has only been recently shown in human
ischaemic kidneys 131. It is possible that inactive
Correspondence: Dr F. Soubrier, INSERM U 36,
17 rue du Fer-a-Moulin,75005 Paris, France.
renin represents a pro- form of renin. In this case
one could expect to find it in malignant tissues in
high concentration and we describe here a
pulmonary renin-secreting tumour containing a
large inactive renin fraction.
Materials and methods
Tumour
Tumour tissue was obtained from an anaplastic pulmonary adenocarcinoma obtained
90 min after death and kept frozen at -30°C.
The patient died from malignant hypereninaemic
hypertension and metastatic spread of the cancer.
All clinical details and pathological studies have
been described elsewhere 141. The tissue was
thawed and immediately homogenized in phosphate buffer (0.1 mol/l; pH 7.5) with the following
protease inhibitors: EDTA 2 mmol/l, N-ethylmaleimide 10 mmol/l, phenylmethylsulphonylfluoride (PMSF) 2 mmol/l, sodium tetrathionate
5 mmol/l, di-isopropylfluorophosphate 50 pmolll.
The homogenate was centrifuged at 30 000 g for
45 min at 4°C and the supernatant was kept.
Renin assays
Renin enzymatic activity was determined by
measuring the amount of angiotensin I (ANG I)
generated. Tumour extract was incubated with
human angiotensinogen for 30 min at pH 5-7,
37"C, in the presence of EDTA 50 mmol/l,
PMSF 2.8 mmol/l in a final volume of 500 pl.
Renin substrate (200 pmol/assay) was a reninfree human substrate prepared by the method of
Guyene et al. 151. ANG I produced was
measured by radioimmunoassay 161. Enzymatic
activity was calibrated in Goldblatt units (G.U.)
against standard MRC human renin.
300s
F. Soubrier et al.
Activation procedures
AcidiJcation. This was performed by dialysing
tumour extract for 24 h against citrate/phosphate
buffer (0.1 mol/l, pH 3.3), followed by 24 h
dialysis against phosphate buffer (0.1 mol/l, pH
7-4). Neutral dialysis was performed by a 48 h
dialysis against phosphate buffer (0- 1 mmol/l,
pH 7.4). In some experiments; benzamidine/HCl
(Sigma) (10 mmol/l) was added. The degree of
activation was measured in comparison with an
aliquot of tumour extract kept frozen.
Pepsin activation. This was performed by the
method of Morris [71, modified as follows: 5-30
p-G.U. of tumour renin was incubated at 37OC
for 5 min with 2.5 pg of pepsin (Sigma) in a final
volume of 50 pl in citrate (0- 1 mol/l)/phosphate
(0.2 mol/l) buffer, pH 4-7, containing 1 mg of
bovine serum albumin/ml. Pepsin was inactivated by raising the pH to 7 . 5 for 15 min at
20°C with 100 p1 of Tris/HCl buffer (0-1 mol/l,
pH 8.4) and renin activity was assayed.
Trypsin activation. The method of Takii et al.
I81 was used: 5-30 p-G.U. of renin was incubated
at 4OC for 15 min with 1 pg of trypsin (Sigma) in
50 p1 of phosphate buffer (0.1 mol/l), pH 7.5,
containing 1 mg of bovine serum albumin/ml.
Incubation was stopped by adding 100 pg of
soybean trypsin inhibitor (Worthington) in 100 pl
of citrate phosphate buffer (0.1 mol/l, pH 5.7)
and renin activity was measured.
Results and discussion
Tumour renin content
The renin enzymatic activity of the tumour
was 10.4 G.U./g of tissue. The concentration of
renin in this tissue is at least a hundred times that
of the normal human kidney [91. It is comparable
with the high renin content found in juxtaglomerular cell tumours [lo] and with the renin
concentration found in the few other cases of
ectopic renin tumours 1 11. Renin concentration
was also measured by direct radioimmunoassay.
There was a strict parallelism between the
standard curve established with pure human renin
or MRC standard human renin and serial
dilutions of the tumour extract, indicating
immunological identity between the ectopic
tumour renin and standard renins. The renin
concentration found by direct radioimmunoassay was 23 G.U./g of tissue.
The discrepancy between the renin enzymatic
activity and the renin content found by direct
radioimmunoassay could be due to either (1) the
presence of immunoreactive fragments of renin
devoid of any enzymatic activity or (2) the
TABLE1 . Activation of inactive turnour renin by different
procedures
Renin activity
(pg of ANG I h-l
Acid dialysis
Neutral dialysis
Pepsin
Trypsin
ml-l)
Before
After
Activation (96)
250
250
313
210
492
443
97
71
69
57
528
328
presence of an inactive form of renin in this
tumour tissue. Similar discrepancies have also
been observed in studies on plasma and amniotic
human renin, where the values obtained by direct
radioimmunoassay are always higher than those
determined by enzymatic assay, owing to the
presence of inactive renin [51. Further studies
were therefore carried out to determine whether
inactive renin also existed in this tumour.
Activation of inactive renin
Several procedures have been used for the
activation of inactive renin. Results are summarized in Table 1.
Acid dialysis was used initially by Lumbers [ 11
to activate human amniotic renin and subsequently by many others for plasma renin studies.
In the present study such procedures gave a
100% activation. Control experiments included
dialysis at neutral pH for 48 h. Surprisingly there
was an activation of inactive renin in these
conditions which was slightly less than that
observed during acid dialysis. Benzamidine, a
potent inhibitor of the serine proteinases, completely abolished this neutral activation but not
the acid activation. It is therefore possible that
acid and neutral activations involve different
mechanisms: the latter might imply the action of
a neutral serine protease. These results can be
compared with those obtained in normal kidney
and kidney turnour extracts: activation of normal
human cadaver kidney has never been reported.
There is only one report of an acid-dialysis
activation in a case of renal tumour [ 121 and one
in a case of Wilms’ turnour [ 131. In plasma, renin
is easily activated by acid dialysis but also at
neutral pH by cryoactivation. The level of this
activation is two- to three-fold lower than that
obtained by acid activation but it can also be
prevented by a serine proteinase inhibitor [ 141.
Proteolytic treatment also activated tumour
renin. However, to demonstrate such an activation it was necessary to optimize the
concentrations of the proteolytic enzymes used.
Indeed the high trypsin concentrations used for
Renin secreted by pulmonary adenocarcinoma
activating inactive human plasma and amniotic
renin decreased renin enzymatic activity, probably by extensive proteolysis of the enzyme. This
discrepancy between the doses used to activate
renin in tissues, compared with that used in
biological fluids, might be due to the presence of
endogenous trypsin inhibitors in the latter. The
fact that activation obtained by proteolytic
treatment did not reach the level of acid dialysis
activation might be due to a slight destruction of
the enzyme coincident with its activation.
In this tumour tissue a large fraction of renin
(about 50%) is therefore in an inactive form and
can be activated by various procedures. Its
presence was suspected because of a discrepancy
between the indirect (enzymatic) and direct renin
radioimmunoassay. The presence of this inactive
enzyme in ectopic tumour tissue, as well as its
immunogenicity being similar to that of normal
renin, points towards a relationship between
ectopic renin and renal renin. Other studies
performed with this ectopic enzyme have shown
similar physicochemical properties to that of the
native enzyme (F. Soubrier et al., unpublished
work). Finally it is interesting to note that the
percentage of renin in the inactive form is much
higher in this tumour than in the normal kidney
(15%) [31. This might be due to a lower content
of proteolytic enzymes in the tumour tissue when
compared with the kidney and(or) to the high
level of production of the enzyme in an inactive
form in tumour tissue, as has been shown for
several prohormones [151.
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