Wilson T. Beraldo

Angiotensin-ll-lnduced Angiogenesis in Sponge Implants in Mice

Stimulators of angiogenesis hold potential in promoting the development of collateral circulation in ischaemic tissue and accelerating would healing, but promote pathological vasoformation in angiogenesis-dependent diseases (solid tumours, atherosclerosis). The renin-angiotensin system is implicated in both beneficial angiogenesis and pathological vascular growth. We investigated the angiogenic activity of angiotensin II (AII) in a sponge implant model in mice; this peptide enhanced angiogenesis, as well as glycosaminoglycan (GAG, chondroitin sulfate proteoglycan) and protein synthesis in sponge matrix in mice in a dose-dependent fashion. Extensive angiogenesis was achieved with AII (1 microgram), which gave no significant increase in wet weight and protein and only a small effect on GAG. In the implants treated with AII (2 micrograms) no further increase in angiogenesis was observed, whereas a marked effect was shown in wet weight (326 +/- 15 vs. 424 +/- 27 mg), total protein (18 +/- 1 vs. 25 +/- 1 micrograms/ww) and GAG (98 +/- 10 vs. 160 +/- 13 ng/ww). The local blood flow has been determined by measuring the washout rate of 133Xe injected into the implants, correlated with histological evidence of vessel growth. This model of angiogenesis has allowed sequential studies of fibrovascular tissue infiltration simultaneously with histological and biochemical parameters of angiogenesis.

Effect of purified scorpion toxin (tityustoxin) on the pancreatic secretion of the rat

Intravenous injection, in anesthetized rats, of a single dose of purified scorpion toxin (tityustoxin, TsTX), obtained from the venom of the Brazilian scorpion Tityus serrulatus, causes a striking increase in flow rate, protein content, kallikrein and amylase activities of the pancreatic juice. The flow rate and protein content of the juice remain significantly higher than in control rats, for at least one hour, whereas the kallikrein activity returns to control values 30 min after tityustoxin injection. Sub-diaphragmatic bilateral vagotomy does not prevent the pancreatic secretion induced by tityustoxin; moreover, vagotomy potentiates the flow rate and kallikrein secretion produced by the toxin. Pre-treatment of the rats with atropine blocks the pancreatic secretion evoked by tityustoxin. It is suggested that the pancreatic secretion induced by tityustoxin is due to actions of acetylcholine, released from postganglionic nerve fibers, on muscarinic receptors. The mechanism by which vagotomy potentiates the pancreatic secretion evoked by tityustoxin is under investigation.

Comparative study of the action of purified scorpion toxin (tityustoxin) on the submandibular and parotid glands of the rat

Abstract Stimulation of the salivary glands by purified scorpion toxin (tityustoxin) evoked flow of saliva and sodium, potassium and kallikrein secretion. Kallikrein secretion from the submandibular gland was up to 150 times greater than from the parotid gland. The parotid gland secretes 20–40 times more sodium than the submandibular gland. Tityustoxin is more effective in eliciting salivary flow in the parotid than in the submandibular gland and potassium secretion seems dependent on salivary flow in both glands. The mechanism of action of tityustoxin involves adrenergic and cholinergic receptors. Tityustoxin may be a suitable tool to study the physiology and pharmacology of the salivary glands.

Distribution of tonin- and kallikrein-like activities in rat brain.

Tonin- and kallikrein-like activities were investigated in different regions of the rat brain. The highest values of specific tonin activity, expressed as picomoles of angiotensin II liberated per minute per milligram of protein, were found in the neurohypophysis (359 +/- 190) and in the archicerebellum (200 +/- 68). The highest level of total tonin activity (picomoles of angiotensin II liberated per minute) was observed in the archicerebellum (902 +/- 308) which retained 97% of total tonin activity of whole cerebellum. Tonin activity was not detected in the cortex of cerebellum and in the choroid plexus. Low to intermediate values of specific (1.09 +/- 0.33 to 5.32 +/- 2.37) and total (1.38 +/- 0.55 to 93.00 +/- 49.30) tonin activity were observed in adenohypophysis, cerebellar nuclei, hypothalamus, thalamus, midbrain, pons, medulla and neurohypophysis. The lowest values of specific (0.11 +/- 0.05) and total (0.69 +/- 0.31) activities were observed in the hippocampus. Kallikrein-like activity was expressed as picomoles of p-nitroaniline liberated per minute per milligram of protein. No activity was detected in the neurohypophysis. For other regions, the values of the specific activity ranged between 72 +/- 18 and 282 +/- 14 except for the choroid plexus which was 5 +/- 2. The total kallikrein activity was also homogeneous ranging from 330 +/- 100 to 1870 +/- 112. For the choroid plexus and adenohypophysis the total kallikrein activity was 2.0 +/- 0.8 and 27 +/- 11, respectively.

Rat uterine contraction by kallikrein and its dependence on uterine kininogen.

Smooth muscle responses to kallikrein (EC 3.4.21.8) are generally considered to result from kinin formation. In the present study, this premise was reexamined with respect to the isolated rat uterus. Rat submandibular gland kallikrein produced contractions of the rat uterus but the contractions disappeared after successive additions of the same dose of the enzyme to the preparation. Kallikrein-induced rat uterine contractions as well as bradykinin-induced contractions were enhanced by rat submandibular gland bradykinin potentiating factor. The incubation of kallikrein with rat uterine extract in the presence of a kininogen-depleted rat uterus produced kinin which elicited the uterine contraction. An extract from uterine horns previously depleted of kininogen was prepared. Incubation of this extract with kallikrein in a bath containing a kininogen-depleted rat uterus did not evoke uterine contraction. The incubation of four rat uterine horns with kallikrein in the presence of a uterine horn previously depleted of kininogen elicited contractions of the depleted uterus. These results suggest that the contraction produced by kallikrein involves kinin release from the uterus.

Discovery of Bradykinin and the Kallikrein—Kinin System

Publisher Summary This chapter provides an overview of the kallikrein (KLK)–kinin system. The chapter highlights the discovery of the physiological and pathological roles of kinins, showing their involvement in vasodilation, regulation of blood pressure, inflammation, and the production of pain. Thus, through the knowledge gained from these pioneering basic studies, the actions of kinins are unveiled, leading to the understanding of many diseases associated with kinin formation. The existence of the KLK–kinin system was first disclosed nearly 50 years ago when a series of coincidental discoveries by two independent groups came together revealing the major biological roles of the system. Werle found that KLK preparations from human urine elicited contractions in isolated dog intestine. Thus, KLK had been shown to possess two activities: induction of hypotension in vivo and contraction of isolated intestinal smooth muscle.

Tonin and Kallikrein-Kinin System

The kininogenase activity of tonin has been demonstrated by Ikeda and Arakawa, 1984. Tonin of the rat submandibular gland contracts the rat uterus independent of addition of the substrate. On repetition, the same dose of enzyme elicited desensitization. When a double dose was used the contraction again occurred. After desensitization to tonin the contraction to kallikrein was reduced about 80% of the control. The desensitization to kallikrein lightly reduced the contraction to tonin. When the muscle was desensitized to trypsin tonin did not evoke contraction. These experiments suggest the presence of two different substrates in the uterus, one more specific to kallikrein and the other for tonin. The experiments with the parallel uterus preparation strongly suggest release of kinin in the process of contraction of the uterus by tonin.

Effect of Isoproterenol on the Kallikrein Content of the Submandibular Gland of Rats

The influence of IPR on the kallikrein content of the submandibular gland of 11, 21, 62 and 90 day old rats was investigated. The appearance of acini in IPR treated rats was associated with an increase in the kallikrein content of the gland in 11 day old rats. The decrease of kallikrein in the gland of 21 to 90 day old animals was attributed to the maturation of the autonomic nervous system and the mechanism of glandular secretion.

Kallikrein-induced rat uterus contraction is dependent on kinin release.

Glandular kallikrein (EC.3.4.21.8) releases lisyl-bradykinin (kallidin) from kininogen substrates by limited proteolysis (Chao et al, 1981). The responses of the smooth muscle to kallikrein are attributed to kinin formation and a subsequent peptide-receptor interaction (Barabe et al,1977; Fritz et al, 1979; Odya and Goodfriend, 1979). Beraldo et al.(1966) reported that rat urinary kallikrein induced rat uterine contractions in the absence of added kininogen. Nustad and Pierce (1974) suggested that uterine contractions elicited by rat urinary kallikrein depend on kinin liberation from some uterine kininogen. Beraldo et al.(1976) demonstrated the presence of kininogen in the rat uterus. Chao et al.(1981) reported that rat glandular kallikrein can cause the contraction of isolated rat uterus independent on kinin formation. As can be seen, the mechanism of kallikrein-induced uterine contraction was uncertain. We used pure rat submandibular gland kallikrein in order to explain this mechanism.

Possible Relationship between Salivary Kallikrein and Water-Na-K Secretion Stimulated by Tityustoxin (TsTx)

Intravenous injection of purified scorpion toxin (TsTx) brings about the appearance of salivary flow with high levels of Na and K and kallikrein in the saliva of the rats. In experiments performed in vivo a positive correlation between Na and partially with K and kallikrein was observed in parotid and submaxillary saliva. After ouabaine, saliva K was increasing, whereas Na decreased, and a parallel decrease of kallikrein was observed. Adrenergic and cholinergic blocking drugs also showed a marked reduction in the output of Na (atropine), and partially of K (atropine or phentolamine), with a correlation in theoutput of kallikrein per min. The results of the present investigations support the hypothesis that salivary kallikrein-kinin system is probably involved in salivary control of Na, partially K and water secretion.