Department

of

Pharmacology, Leo Pharmaceutical Products, 2750 Ballerup, Denmark

KININASE INHIBITION BY GLUCAGON

By

Uffe Bang Olsen ABSTRACT In vitro glucagon (1\p=n-\100\g=m\g/ml) has been shown to inhibit the degradation of bradykinin by kininases present in kidney microsomes and erythrocytes. Glucagon was less active on kininases present in plasma and was inactive on purified pancreatic carboxypeptidase B. In conscious dogs the intravenous infusion of glucagon (10 \g=m\g/min) increased urine flow (160 %) and urine kinin excretion (130%) and decreased urine kallikrein excretion (14 %). It is suggested that in vivo inhibition of kininases might contribute to the biological effects of glucagon.

Glucagon in pharmacological doses produces vasodilation (Kones 8c Phillips 1971), and it has been suggested that glucagon should be used therapeutically to improve regional blood flow e. g. in kidneys (Levy 1975) and liver (Darle et al. 1976). The mechanism of the vascular effect is unknown. In kidneys, however, the vasodilation is associated with an enhanced prostaglandin activity (Olsen 1977) and natriuresis (Stowe 8c Hook 1970; Levy 8c Starr 1972; Olsen 1977). Kidney kinins, bradykinin and kallidin, also increase renal blood flow and sodium excretion (Barraclough 8c Mills 1965; Heidenreich et al. 1964; Webster 8c Gilmore 1964), and release prostaglandins (McGiff et al. 1972). The present study was therefore performed in order to investigate a possible relationship between glucagon and the kallikrein-kinin system, and it is shown that glucagon inhibits kininase activity. METHODS

In vitro

Preparation of kininase sources Dog kidneys cortical tissue

were was

perfused

blood-free with ice-cold Krebs-Henseleit solution. The 10 seconds

minced, weighed and homogenized (Ultra Turrax) for 552

in 20 vol. 50 mM KH2P04/NaOH buffer, pH 7.4. The microsomal fraction containing the membrane vesicles was sedimented from the supernatant by centrifugation at 105 000 g for 60 min after initial centrifugation at 10 000 g for 20 min. The micro¬ somal fraction was Iyophilized and stored at -25°C. When use 10 mg microsomes was suspended in 2 ml 0.1 m Tris, pH 7.4. Plasma was separated by centrifugation of dog venous blood collected on heparin (10 jiXJ/ml). Erythrocytes were then washed twice with Tyrode's solution, and were haemolysed in 10 vol. distilled water. Purified carboxypeptidase B (400 U/ml) was purchased from Merck (Darmstadt).

Enzyme

assays The kinin substrate contained 100 ng of bradykinin (Sandoz) per ml 0.1 M Tris, pH 7.4. Kininase activity was assayed by incubating samples of cortical microsomes (10 ¡a\ 34 pig of protein), plasma (50 jil), haemolysate (50 «1 5 jig of erythrocytes) or purified pancreatic carboxypeptidase B (2 mU) with 1 ml substrate at 22°C. The inactivation of the peptide was followed on the isolated rat uterus (Olsen Se AhnfeltRonne 1976), and the inhibitory effect of 1-100 ,«g/ml glucagon (NOVO) on kininase activity was tested. =

~

In vivo Ten pig/min glucagon was infused iv for 30 min in 6 normal conscious dogs, and urine was collected via an indwelling bladder catheter as reported in previous experi¬ ments (Olsen 1977). Urine kallikrein activity was determined by the micromethod of Marin-Grez et al. (1972) as previously described (Olsen Se Ahnfelt-Ronne 1976). Urine kinin excretion was determined by bioassay on dog hind limb blood flow after rapid and cold n-butanol extraction and purification as described by Nasjletti et al. 1975).

n T

5' 10'

2' 5' 10'

2'

5' 10'

2' 5' 10'

Bradykinin ng/ml 0,5 q2 Q1

Fig. 1. bradykinin by dog kidney microsomes and the inhibition by glu¬ cagon 1 /

Kininase inhibition by glucagon.

Department of Pharmacology, Leo Pharmaceutical Products, 2750 Ballerup, Denmark KININASE INHIBITION BY GLUCAGON By Uffe Bang Olsen ABSTRACT In vi...
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