T B Orstavik

Immunohistochemical localization of kallikrein in human pancreas and salivary glands.

The localization of kallikrein in human exocrine organs was studied with a direct immunofluorescence method. In the submandibular and parotid salivary glands, kallikrein was found apically in the striated duct cells whereas it was absent from the main excretory ducts or present only as a weak luminal rim. Kallikrein was not found in the acinar cells or in cells of the intercalated ducts. In the pancreas, kallikrein-specific fluorescence was seen in the granular portion of the acinar cells, whereas the islets of Langerhans and ductal cells were unstained.

Immunohistochemical localization of tonin and its relation to kallikrein in rat salivary glands.

Tonin and kallikrein are serine proteases present in high concentrations in the submandibular gland of the rat. These enzymes release the vasoactive peptides angiotensin II and lysyl-bradykinin from the precursors angiotensinogen and kininogen, respectively. Tonin and kallikrein were purified from homogenates of rat submandibular gland, and antisera against each protein were raised in rabbits. The anti-kallikrein antibody also reacted with tonin, showing partial cross-reactivity between kallikrein and tonin when tested by double immunodiffusion and by immunoelectrophoresis. The anti-tonin antibody did not appear to react with kallikrein in immunodiffusion systems. The cellular localization of tonin was investigated by the indirect immunofluorescence and the peroxidase-antiperoxidase techniques. In the granular tubular cells tonin-specific staining was abundantly present with a granular distribution; in the striated duct cells tonin-specific staining was observed as a thin luminal rim. Tonin was not detected in any other structures of the gland. When the localization of tonin was compared with that of kallikrein, both enzymes were found within the same granular tubular cells. However, more kallikrein than tonin was detected in the striated duct cells. Furthermore, kallikrein but not tonin was found in the ductal cells of the parotid and sublingual glands.

Role of kallikrein in the hypertensive effect of captopril after sympathetic stimulation of the rat submandibular gland.

After cervical sympathetic nerve stimulation, the rat submandibular gland releases a significant amount of kallikrein into the circulation. To determine whether this glandular kallikrein has kininogenase activity (kinin-generating capabilities) in the peripheral circulation and whether it plays a role in blood pressure regulation, we studied the effect of captopril on blood pressure in 48 hour-nephrectomized rats with and without prior sympathetic stimulation of the submandibular gland. Administration of captopril 10 minutes after gland stimulation resulted in a mean blood pressure decrease (A BP) of 43 ± 8.3 mm Hg (P 0.05). To confirm that the effect of captopril was due to a blockage of kinin destruction generated by glandular kallikrein and not to the inhibition of angiotensin II formation, we determined the effect of captopril after gland stimulation in rats pretreated with either IgG from nonimmunized rabbits (normal-IgG), or IgG from rabbits immunized against kinins (antikinin-IgG) or kallikrein (antikallikrein-IgG). Normal-IgG did not significantly alter the hypotensive effect of captopril (A BP —30 ± 7.7; P < 0.01), while pretreatment with antikinin or antikallikrein almost completely blocked its hypotensive effect (A BP —5.8 ± 1.9 and —6.4 ± 0.4, respectively). In the latter two groups, the decrease in BP was significantly smaller (P < 0.001) than in the groups that were not pretreated or in the group pretreated with normal-IgG. These data suggest that, upon adrenergic stimulation of the submandibular gland, glandular kallikrein released into the vascular compartment has kininogenase activity in the peripheral circulation. The kinins released by glandular kallikrein induced hypotension when their breakdown was prevented by the kininase II inhibitor, captopril. These results suggest that kinins may be responsible in part for the antihypertensive effect of captopril in situations in which glandular kallikrein in blood is increased. (C/rc Res 51: 385-390, 1982)

A biochemical and immunohistochemical study of kallikrein in normal and isoproterenol-stimulated rat salivary glands during postnatal development

Abstract The postnatal development of kallikrein-producing cells in the rat submandibular and sublingual salivary glands was followed by immunohistochemistry and by studies of the enzymic and antigenic activity of gland homogenates. Kallikrein was absent in the new-born rat, but appeared during the first week of life in the immature duct cells of both glands. Increased concentrations of extractable enzyme paralleled the development of the duct system. In homogenates of the submandibular gland, kallikrein activity increased markedly between the age of 1–2.5 months when the granular tubules developed. Kallikrein-containing granular tubular cells, as shown immunohistochemically, evidently accounted for the high enzyme concentrations found in the adult submandibular gland homogenates. Administration of isoproterenol produced an increase of acinar growth; the accompanying decrease of extractable kallikrein activity in relation to total protein paralleled the measured reduction of the ratio between ducts and acini. At no stage in the normal or drug-induced glandular development was kallikrein demonstrated immunohistochemically in acini or other non-ductal cells. Moreover, its ductal concentration was unaffected by induction of acinar growth. No qualitative or quantitative differences in the production of kallikrein were found to be related to sex.

Effects of different tissue processing methods on the immunohistochemical localization of kallikrein in the pancreas.

Our recently published article (8) on the acinar localization of kallikrein in the human pancreas agrees with our previous study in the rat ( 10) and with observations on the porcine pancreas made by Died et al. (4). An acinar localization is also in accordance with kallikrein quantitations performed on isolated pancreatic acinar and islet cells ( 3 ). Furthermore, the secretion of pancreatic kallikrein parallells that of trypsin (5), which is exclusively derived from the acini. None of these studies have provided evidence for the presence of kallikrein in endocrine cells. By contrast, ole-MoiYoi et al. (6) reported that the localization of kallikrein in the human pancreas was restricted to the B cells in the islets. Being well aware of the various pitfalls in immunohistochemistry, we took great care in characterizing our immunological reagents and in establishing the specificity of the immunofluorescence staining ohtamed in the rat and human pancreas. The discrepancy between our result and that of ok-MYoI et at (6) cannot be explained merely by differences in antilxxly specificity, neitherwith regard to re tiviry towards different antigen epitopes, such as the active enzyme sire, nor with regard to the presence of coormtinating antibodies, as was suggested by c)le-MoiYo. et aL (7). Our antisera were raised against the active enzyme, and the antib y to rat kallikrein was shown to re t equally well with pancreat prokaftikrein tivated enzyme (Il). Moreover, since there re no major differences in the char rerisrics of the isolated human protein used immunogen by the two research groups, we have to assume that xiviry to kallikrein was the principal antibody operating in the nctual immunohi ochemical methods of both laboratories. As a conseq*. nce, at least one of the research groups should have found kallikrein in both acinar and islet cells if it indeed is present in both sites. We, therefore, concluded that the

Radioimmunoassay of Rat Submandibular Gland Kallikrein and the Detection of Immunoreactive Antigen on Blood

Glandular kallikreins are a group of related kinin-forming enzymes (EC 3.4.21.8) present in the major exocrine glands and in the kidney. They are found in the ductal cells of the salivary glands, in the distal tubular cells of the kidney and in the acini of the pancreas (Nustad et al., 1978b). After activation of the glands, kallikrein is found in the secreted fluids such as saliva, pancreatic juice and distal tubular urine. However, evidence indicate that glandular kallikreins may be released to blood during salivary gland stimulation (Nustad et al., 1978b). The substrate for glandular kallikreins is present mainly in blood and formation of the active product lysyl-bradykinin implies that enzyme and substrate combines in the intercellular space or in blood. However, so far glandular kallikreins have not been detected in blood except during acute pancreatitis where an inhibitor-pancreatic kallikrein complex was found (Ofstad, 1970). The establishment of a sensitive radioimmunoassay for glandular kallikreins has allowed us to explore the possible occurrence of glandular kallikreins in blood. Employing antiserum raised against purified rat submandibular gland kallikrein, we demonstrate the presence of immunological crossreacting material(s) in plasma. A preliminary report was presented at the Nordic Micro-circulation group (Nustad et al., 1978a; Orstavik et al., 1978).

Excess antibody immunoassays for rat glandular kallikreins. Measurement of kallikrein from different organs in the presence of cross-reacting antigens.

An immunoradiometric assay has previously been developed for measurement of rat glandular kallikrein. In the present paper, further studies on the specificity and sensitivity of the method are described. Problems of interference of immunologically cross-reacting antigens were overcome by proper preabsorption of the antibody. A method was thus established in which enzymatic activity of the immunoreactive kallikrein could be measured even in the presence of enzymes sharing immunological determinants and substrate specificity with kallikrein. Two variants of the immunoradiometric assay have been evaluated. A simplified version with simultaneous addition of all reagents gave results equal to those obtained in the original assay. A further modification with delayed addition of the solid-phase antibody, gave considerable improvement in assay sensitivity.

An immunohistochemical study of kallikrein in the rat parotid and exorbital lacrimal glands

Abstract In the parotid gland, kallikrein was found by the direct immunofluorescence technique in the cytoplasm of the intralobular and interlobular striated duct cells and as a luminal rim in the main excretory ducts. Kallikrein-specific fluorescence was not found in the exorbital lacrimal gland which lacks striated ducts. The function of glandular kallikreins may be associated with striated ducts or similar structures elsewhere.

INTERFERENCE OF CONVERTING ENZYME INHIBITORS WITH THE KALLIKREIN-KININ SYSTEM

The unstimulated rat submandibular gland releases kallikrein into the circulation. This release is greatly increased by sympathetic nervous stimulation of the gland. We studied the effect of an angiotensin I converting enzyme (ACE) inhibitor (captopril) on blood flow of the unstimulated submandibular gland, and the effect of the ACE inhibitor on blood kinins and blood pressure of rats with prior sympathetic stimulation of the gland. To minimize the effects mediated by inhibition of angiotensin II formation, all rats were nephrectomized 48 hours before the studies. Administration of the ACE inhibitor caused a 2.4-fold increase in the blood flow of the unstimulated gland (p less than 0.001). Pretreatment with kinin antibodies diminished this increase by 86% (p less than 0.001). After sympathetic stimulation, blood flow to the gland increased five times, and kinin output in the venous effluent increased from 19 +/- 13 to 14,400 +/- 8,800 pg/min (p less than 0.001). Despite this conspicuous increase, arterial blood kinins did not change. Administration of the ACE inhibitor after stimulation of the gland resulted in a 10-fold increase in arterial blood kinins and in a pronounced decrease in blood pressure from 100 +/- 5 to 58 +/- 6 mmHg. This decrease was almost completely blocked by antibodies to either kallikrein or kinin. We concluded that the glandular kallikrein-kinin system may participate in the regulation of local blood flow in organs rich in glandular kallikrein such as the rat submandibular gland.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of Glandular Kallikreins and Secretion of Kallikrein from the Major Salivary Glands of the Rat

Glandular kallikreins (E.C.3.4.21.8) are found in major exocrine glands as well as in the kidney. These kinin-forming enzymes are also present in the glandular secretions and preurine, and, they are believed to be synthesized in the respective organs (Nustad et al., 1975). The physiological function of kallikrein in exocrine glands and the kidney is incompletely understood. However, the cellular localization and secretory pattern of kallikrein may give some indication as to the physiological role of kininogenases in these organs. Moreover, such information is also important for designing experiments to study the physiological function of glandular kallikreins. The cellular localization of kallikrein was therefore investigated by means of a direct immunofluarescence technique in the major salivary glands, the pancreas, the kidney, and the exorbital lacrimal gland of the rat. The secreti kallikrein into saliva from the major salivary glands was investigated after various types of gland activation. Some of these results have been published previously (Orstavik et al., 1975; Orstavik et al., 1976; Orstavik and Gautvik, 1977; Orstavik and Gleaner, 1978).