Effects of pulmonary circulation on blood levels of insulin and glucagon in ambulant dogs DUNCAN M. GEDDES, J. P. BLACKBURN, Westminster Hospital, London S WI, United

GEDDES,DUNCAN M.,J. P. BLACKBURN, J.S. BAILEY, AND F. J. MULLER. Effects ofpulmonary circuZation on Mood ZeveZs of insulin and glucagon in ambulant dogs. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 46(3): 593-598,1979.-The effect of the lungs on insulin and glucagon were studied by comparing aortic and mixed venous levels in ambulant dogs with indwelling catheters. In the resting state there was no difference for either hormone. After injection of insulin aortic levels exceeded mixed venous for 10 min; mixed venous levels exceeded aortic for the next 35 min. After injection of glucagon mixed venous levels consistently exceeded aortic. Single-pass studies showed an apparent gain in immunoreactive insulin as compared with [ 14C]inulin and ‘251-albumin during passage through the lung; but no effect on glucagon could be demonstrated. A loss of immunoreactive hormone across the lung might be explained by degradation within the capillary lumen by endothelial cell surface peptidases or uptake onto specific receptors. The apparent gain of immunoreactive insulin across the lung might be due to displacement of immunoreactive insulin from the lung by relatively less immunoreactive exogenous hormone competing for receptor sites or modification of exogenous hormone by the lungs with increase in immunoreactivity. immunoreactivity;

inulin;

lung metabolism

of the lung as a metabolic organ has been emphasized in many recent reviews (1, 8, 14). Attention has centered largely on short-acting vasoactive substances and, in particular, &hydroxytryptamine, catecholamines, bradykinin, angiotensins, and prostaglandins. Little work has been done on polypeptide hormones with longer circulating half lives. Rubenstein et al. (12) reported a fall in the circulating concentration of insulin during passage through the lungs by comparing mixed venous and arterial levels of insulin in blood taken from patients at cardiac catheterization; Jonsson (6) reported that in two young acidotic diabetics only about 25% of an intravenous injection of insulin could be recovered in arterial serum after one passagethrough the lungs. These in vivo studies were limited in scope and have not to our knowledge been confirmed, although other workers have shown breakdown of insulin by lung homogenates (9). We have therefore investigated the effect of passage through the pulmonary circulation on insulin and glucagon in a series of experiments using ambulant dogs with indwelling catheters. Our study was carried out in three stages: 1) comparison of mixed venous and systemic arterial levels of hormone in the fasting dog; 2) comparison of mixed venous

THE IMPORTANCE

0161-7567/79/0000-0000$01.25

Copyright

J. S. BAILEY, Kingdom

AND

F. J. MULLER

and arterial levels of hormone for 45 min after an intravenous injection of insulin or glucagon; and 3) comparison of arterial levels of insulin with albumin and inulin after a single passagethrough the pulmonary circulation. METHODS

Eleven mongrel dogs weighing 15-23 kg (mean 20 kg) were prepared by insertion of cardiac catheters into the jugular vein, pulmonary artery, and aorta under general anesthesia. Polyethylene 8 French gauge catheters were inserted into the pulmonary artery (or right ventricle) and aorta and a Portex nylon 8F catheter was placed in the jugular vein. The dead space of the pulmonary artery catheter was 2.0 ml and of the aortic catheter 1.2 ml. The catheters were brought out at the back of the neck, filled with heparin (1,000 U/ml), and capped. The animals were allowed to recover and were subsequently given subcutaneous heparin 500 U twice daily during the time the catheters were in place. Catheters could be maintained in this way for periods up to 6 wk. Studies were performed once or twice a week after checking hemoglobin and hematocrit values. During the experiment the animal was heparinized 3,000 U intravenously and the catheters were flushed with heparinized saline as required. No other drugs were administered. Position of the catheters was confirmed radiographically or by presssure measurements. Measurements of transpulmonary circulation time and recirculation time were made. A bolus of indocyanine green (Hynson, Westcott and Dunning) was injected into the pulmonary artery and blood was sampled from the aorta at 0.8 ml/s and passed through a Gilford densitometer (model 1031R). Transpulmonary circulation time was estimated from appearance time of the dye; the time when recirculation occurred was determined following semilogarithmic replot of the washout phase of the indicator-dilution curve. Nine experiments were performed on three dogs. Experiment

I

Six-milliliter samples of mixed venous and aortic blood were withdrawn at the same time from conscious ambulant fasting dogs. To ensure that the equivalent blood was sampled on each side of the lung, the aortic sample was delayed 2-3 s after the mixed venous to allow for passageof blood through the lungs. Allowance was made for the dead space in the catheters by synchronous

0 1979 the American Physiological Society

Downloaded from www.physiology.org/journal/jappl at Midwestern Univ Lib (132.174.254.157) on February 18, 2019.

593

594

GEDDES,

withdrawal of dead space blood in the 5 s before sampling. Fourteen paired samples for insulin assay and eight paired samples for glucagon assay were taken from six dogs. Experiment

2

An injection of hormone was made into the venous catheter and flushed in with 8 ml normal saline. Timed samples of mixed venous and aortic blood were then withdrawn as described above. Three doses of soluble bovine insulin (Weddell 40 U/ml) were given: 0.04 U/kg (3 expt, 2 dogs), 0.07 U/kg (5 expt, 3 dogs), and 0.15 U/ kg (6 expt, 4 dogs). One dose of glucagon (0.5 ,ug/kg) was given (5 expt, 3 dogs). In 10 of these experiments the venous sampling catheter tip was in the pulmonary artery and in nine in the right ventricle. Experiment

3

The effect of single passage through the lung was investigated using insulin. A solution of soluble bovine insulin (Weddell 40 U/ml); [14C]inulin (( hydroxy[ 14C]methyl)inulin, Radiochemical Centre, Amersham), and ‘251-albumin (iodinated 1251-human albumin injection BP, Radiochemical Centre) was made up in human plasma protein fraction BP (Lister Institute, Elstree) to give insulin 190 mu/ml, [14C]inulin 2.5 ,&i/ml, and ‘““I-albumin 0.5 pCi/ml. This solution was stored at -2OOC as 2ml aliquots. For each experiment 0.5 ml of this solution was injected into the pulmonary artery catheter and then flushed into the circulation as a rapid bolus with 8 ml of normal saline. Immediately before each injection, 10 ml of aortic blood were taken for analysis and then from the moment of injection aortic blood was sampled at a constant rate of 1 ml/s for 12 s. This 12-s sample represents the majority of the outflow of the injected solution from the lungs after one pass through the pulmonary circulation, and was timed to coincide with the indocyanine green dye-dilu tion curves up the time of recirculation. Two dilutions of the injection solution were made in the aortic blood taken prior to the experiment and were used as standards. Insulin, [ “C]inulin, and ‘251-albumin were estimated in the injection solution .s, the standard dilutions, and the aortic blood sampled before and after the injection. This experiment was performed eight times on two dogs for insulin and four times on two dogs for glucagon. In a variant of this experiment a solution containing 1251-insulin (1.3 &i/ml) and ‘311-albumin (1.3 pCi/ml) was made up in plasma protein fraction 10% in normal saline and was injected as a bolus into the pulmonary artery. Sequential l-s samples of aortic blood were then taken for the next 15 s by moving the end of the aortic catheter from one tube to the next at l-s intervals. In this way, first-pass dilution curves for insulin and albumin could be constructed. This experiment was performed twice on two dogs. Blood samples were centrifuged for 5 min at 3,000 rpm immediately after collection, separated and stored at -2OOC.

Assay

BLACKBURN,

BAILEY,

AND

MULLER

Methods

Insulin. Fifty microliters of sample or standard (661 304) were incubated with 500 ,pl 0.2% albumin-borate buffer, 100 ~1 ‘251-insulin, and 100 ~1 guinea pig antiinsulin serum (Guilhay HP/D/4) at 4°C for 24 h. Rabbit anti-guinea pig serum (100 ,ul) and normal guinea pig serum (100 ~1) were then added and incubation was continued for a further 24 h. Separation was achieved by centrifugation at 2,400 rpm at 4OC for 1 h. The precipitate was washed with 500 ~1 of 0.9% saline and recentrifuged. This precipitate was counted for 1 min in an automatic gamma counter. This system gives a 50% zero binding with a nonspecific binding of 6%. The intra-assay coefficient of variation is 9.8%. The recovery of known amounts of insulin in this system approaches 100%. GZucagon. Two hundred microliters of sample or standard (MRC pancreatic glucagon), 100 ~1 rabbit anti-glucagon solution, and 100 ~1 of tyrosylated 1251-pancreatic glucagon were incubated with 400 ~1 of buffer (0.05 normal veronal buffer pH 8.0 containing 5% human glucagon-free plasma and 1% aprotinin) at 4OC for 5 days. Charcoal (20 mg, as 10% suspension in dextran) was added and separation achieved by centrifugation at 4°C for 1 h (2,000 rpm). Supernatant and precipitate were counted in an automatic gamma counter. The incubation of 5 ,ul undiluted glucagon antibody with this system results in 87% binding of the label. were esRadioactivity. ‘251-albumin and ‘“‘I-albumin timated in l-ml serum samples by counting of the appropriate isotope in a Wallace gamma counter GTL 300-500. Serum [14C]inulin was measured after precipitation of protein with 10% trichloroacetic acid in an Intertechnique SL30 liquid scintillation counter using 2,5-bis[5’-tert-butyl-benzoxazolyl-(2’)lthiophene (BBOT, Sigma Chemical Co.) as scintillant. Total immunoprecipitable 1251-insulin was assayed by a standard double-antibody technique described by Morgan and Lazarow (10) as modified by Sonksen et al. (13), except that charcoal-treated dog serum was used in place of human serum and guinea pig anti-insulin serum was used in excess. RESULTS

Indocyanine

Green Dye Curves

In nine experiments mean t SD for transpulmonary circulation time was 2.6 t 0.8 s, and recirculation time was 11.8 t 1.0 s. Experiment

1

Mean levels of insulin and glucagon in mixed venous (V) and aortic (a) blood are shown in Table 1. There were no significant differences. Experiment

2

Insulin. Insulin levels in mixed venous and aortic blood are shown in Table 2 according to insulin dose and time after insulin injection. The insulin decay curve following injection of 0.15 U/kg is shown in Fig. 1. Mixed venous

Downloaded from www.physiology.org/journal/jappl at Midwestern Univ Lib (132.174.254.157) on February 18, 2019.

INSULIN,

GLUCAGON,

AND

1. Hormone and aortic blood -

.~___.----_____ Mixed

n

Values

14 8

are means

595

CIRCULATION

levels in mixed venous

TABLE

Insulin, mU/l Glucagon, fmol/l

PULMONARY

Venous

(V)

---~

Aortic

10.75 t 1.63 17.5 t 2.4

Mixed VenousAortic Diff (V - a)

(a)

10.21 t 1.62 16.4 t 1.6

0.54 t 0.81 1.1 t 1.2

2. Insulin levels in mixed venous and aortic blood after rapid intravenous injection of insulin ~ __-. -- ~~~~~ _ ___--

TABLE

Min after Injection

n

Mean V

0.04

2 3 5 7 10

3 3 3 3 3

167 122 91 62 24

1 1.5 2 3 5 7 10

5 4 5 4 4 4 4

3 5 7 10 15 20 30 45

5 5 5 5 6 5 5 5

V - a, Mean f SE

(V - a)/S;, x 100, Mean

185 138 100 65 30

-18 t 2.0 -16 k 6.9 -9 t 4.3 -3 t 5.3 -6 t, 1.8

-11 -12 -11 -6 -28

368 326 238 182 124 91 63

413 338 238 191 133 90 63

-45 -12

404 303 212 153 83 49 27 15

460 315 229 154 78 49 21 12

-56 -12 -17 -1

Mean

a

3

500

0.07

0.15

n (V -

Experiment

The ratio of insulin to inulin and insulin to albumin measured in the injection solution and in the aortic blood collected for 12 s after bolus injection are shown in Table 4. The ratio in the postinjection sample is calculated from the increase in the level in the aortic blood over the level measured in the sample taken immediately before

t, SE.

Insulin Dose, U/kg

malized values (V - a)/V is significant for timed samples up to 15 min (P < 0.05). Again the later samples when V is low show a wide scatter of (V - a)/V. The glucagon decay curves are shown in Fig. 2.

a)/v

All pairs

V-a Insulin

All pairs levels

are given

O-10 min 15-45 min 15-45 min in mu/l.

65 21 21 V, Mixed

38.4 30.7 23.2 7.6 12.2 8.8 1.2

-14 -4

1 0

t t t t -+ t, t

33.6 11.1 26.9 4.6 2.3 2.1 2.2 2.8

-17 -5 -11 -1

5 0 6 3

t t t t t t I!I k

0 -9 -9

Mean

f SE

-7.6 t 2.09 9.7 t, 4.68 3.2 t 1.19 venous;

Mixed

-

Aortic

venous

400

0 -6 -5

300

4 0 Insulin mU/l

200

6 0 20 13 100

P