J. A. V. Simson

Kallikrein localization in rodent salivary glands and kidney with the immunoglobulin-enzyme bridge technique.

Kallikrein has been localized in rodent kidney and salivary glands by means of an immunoglobulin-enzyme bridge technique. In sections of kidney, anti-kallikrein antibodies bound to the apical region of certain distal tubule segments in the cortex, to reabsorption droplets of proximal convoluted tubules, and to certain duct segments in the papilla. In salivary glands of both male and female rats and mice, and apical rim of most striated duct cells of submandibular, parotid and sublingual glands and granular tubules of submandibular glands exhibited immunoreactivity. Granular intercalated duct cells in female submandibular glands also displayed immunostaining for kallikrein. Phenylephrine administration resulted in loss of immunoreactive granules from the granular convoluted tubule cells of male mouse submandibular gland. This response was paralleled by a biochemically demonstrable decrease in kallikrein-like tosylarginine methyl ester (TAME) esterase activity.

Immunocytochemical localization of tissue kallikrein in brain ventricular epithelium and hypothalamic cell bodies.

A specific monoclonal antibody against rat tissue kallikrein was used as the primary antibody for indirect immunoperoxidase staining of rat hypothalamus. Kallikrein was localized in the epithelial cells (ependyma) lining the third ventricle as well as in cell bodies of arcuate, supraoptic, paraventricular, and ventromedial nuclei.

The effects of reserpine on the ultrastructure and secretory responses of rat exocrine pancreas

Abstract The effects of chronic reserpine administration on rats mimic in several respects the exocrine dysfunction observed in cystic fibrosis. This drug treatment has been proposed as a model for the human disease. In cystic fibrosis, the pancreas is usually a major organ involved pathologically. The present study was designed to investigate the fine-structural morphology and secretory responses of the pancreas of rats treated chronically with reserpine. Reserpine treatment resulted in increased storage of zymogen granules in pancreatic acinar cells and an apparently reduced ability to discharge these granules following stimulation with cholecystokinin. In addition, granule storage may have produced feedback inhibition on the protein synthesizing machinery as manifested by a slight reduction of rough endoplasmic reticulum. Many acinar cells also had autophagic bodies, suggesting the degradation of excess secretory material. Cholecystokinin stimulation of both control and reserpine-treated rats resulted in the appearance of large vacuoles containing myelin figures and granular material, numerous autophagic bodies (cytosegresomes), and a slight decrease of rough endoplasmic reticulum in pancreatic acinar cells. Secretin stimulation did not cause large vacuole formation, but did cause cytosegresome formation in acinar cells. Large vacuoles in intralobular ducts were noted after reserpine treatment, but were also present after stimulation of untreated pancreas with cholecystokinin.

Localization of copper-zinc superoxide dismutase in the endocrine pancreas

Immunoelectron microscopy and cellular fractionation on sucrose density gradients have been used to examine the intracellular distribution of copper-zinc superoxide dismutase (SOD) in the canine and rat endocrine pancreas. Using rabbit anti-canine copper-zinc SOD as the primary antiserum, immunostaining in canine beta and non-beta islet cells was significantly greater than that in serial sections of the same islet incubated with preimmune serum from the same rabbit. Within the cells, immunostaining was associated with spherical and crystalloid granules as well as with the cytoplasm. Radioimmune assays of cellular fractions or rat islet-cell preparations showed that the granule-rich fraction containing large amounts of insulin was also rich in SOD. This interesting observation of association of SOD with hormone-containing granules suggests that SOD may play a role in protecting these oxidation-sensitive proteins.

Electron Microscopic Immunostaining of Kallikrein in Rat Submandibular Glands

The subcellular localization of glandular kallikrein in ducts and tubules of the rat submandibular gland was determined using a postembedding immunostaining (peroxidase--antiperoxidase) technique on thin sections of Eponembedded tissue. Kallikrein was found in large granules of granular convoluted tubule cells and in small, apical granules of striated duct cells. It was also present in patchy aggregates along the surface of striated duct cells and intercalated duct cells, but not in granules of the latter. Basal dense bodies (lysosomes?) of granular tubules also stained for kallikrein. Absorption of kallikrein antiserum with rat urinary kallikrein, but not with rat urinary esterase A, abolished the specific immunostaining in these sites.

Regulation of Kininogen Gene Expression and Localization in the Lung after Monocrotaline-Induced Pulmonary Hypertension in Rats

Abstract Pyrrolizidine monocrotaline (MCT) from plant seed produces pulmonary endothelial cell injury, pulmonary hypertension, and inflammation in rats, providing a useful animal model for studying progressive pulmonary vascular disease. Kininogen is the precursor of proinflammatory kinins and may also exert anti-inflammatory actions by inhibiting cysteine proteinases. Given the potential roles of kininogen in vascular injury and inflammation, we have investigated the regulation of kininogen gene expression in the MCT-induced pulmonary hypertensive rat model. Sprague-Dawley rats, in groups of six, were given a single subcutaneous injection of monocrotatine (60 mg/kg body wt) and sacrificed 10 and 20 days later. Northern blot hybridization using a kininogen cDNA probe showed kininogen gene expression in the liver, lung, and kidney. MCT treatment induced a time-dependent increase in kininogen mRNA levels, whereas it reduced rat α1-antitrypsin and kallikrein-binding protein mRNA levels in the liver. Similarly, kininogen mRNA levels were low in the normal lung and were increased 7.5- and 13.7-fold, respectively, after MCT injection for 10 and 20 days. Immunoreactive kininogen levels in perfused liver and lung extracts of rats receiving MCT injection increased up to 20-fold, as measured by a T-kininogen radioimmunoassay. Western blot analyses showed that a 68-kilodalton immunoreactive kininogen increased in the serum and lung extracts of MCT-treated rats compared to those in the control rats. In control rats, immunostaining for kininogen in the lung was most marked in venous endothelial cells and alveolar macrophages. After MCT treatment, staining for kininogen increased dramatically throughout the lung tissues, often covering the epithelial surfaces of alveoli and bronchi. The present studies have shown that the toxin MCT altered the synthesis and distribution of pulmonary kininogen and suggest that the kininogen/kinin system may be associated with the pulmonary vascular injury, remodeling, and inflammation seen in this animal model.

Co-localization of a kallikrein-like serine protease (arginine esterase A) and atrial natriuretic peptide in rat atrium.

Atrial natriuretic peptide (ANP) is stored in atrial granules primarily as a larger molecular weight precursor (pro-ANP), which is believed to be rapidly converted to an active peptide of 28 amino acids during or shortly after secretion. A tissue kallikrein-like serine protease has been suggested as a potential processing enzyme. In the present immunocytochemical study, using specific monoclonal antibodies, we found that esterase A, a kallikrein-like serine protease, was demonstrable in rat atrial myocytes and in ventricular myocytes, and was capable of cleaving pro-ANP to yield a low molecular weight product. Using colloidal gold immunocytochemistry at the electron microscopic level, we have found esterase A in atrial myocytes, both in granules and in another subcellular site that corresponds to sarcoplasmic reticulum. Double-label electron microscopic immunocytochemical results indicated that esterase A can co-localize with ANP in granules of atrial myocytes.

Unique deposits in mast cells fixed with pyroantimonate osmium tetroxide

Abstract Evidence for a unique cation distribution in mast cells in a variety of tissues was obtained by fixation with a modification of the potassium pyroantimonate-osmium tetroxide procedure of Komnick. Fine antimonate deposits with a heterochromatin distribution were abundant in nuclei of other cell types but were diminished to absent in mast cell nuclei. Nuclei of mast cells usually contained large coarse precipitates which measured 100–150 nm in diameter and were distributed peripherally, as well as less coarse deposits, which measured about 50 nm, and were distributed throughout the nucleus. Cytoplasmic deposits distributed in Golgi vesicles, between granules and at the plasmalemma were observed in some cells. It is suggested that these unusual deposits result from precipitation of antimonate by histamine or by cations whose distribution is affected by the highly ionic materials present in mast cells. Mast cells of man differed from those of rodents in containing cytoplasmic lipid droplets with peripheral antimonate deposits.

Comparison of the distribution of tissue kallikrein and esterase A, a kallikrein-like enzyme, in rat kidney using specific monoclonal antibodies.

Tissue kallikrein (E.C. 3.4.21.35) and arginine esterase A, another closely related, kinin-generating serine protease, have been localized by immunocytochemistry in rat kidney, using monoclonal antibodies that do not crossreact with other kallikrein-related enzymes or with tonin. Kallikrein was present primarily in the apical cytoplasm of the connecting tubule and the cortical collecting duct. Esterase A, on the other hand, was present primarily in the basolateral region of both proximal and distal straight tubules in the outer medulla and medullary rays. In addition, esterase A was demonstrable in distal convoluted tubules and, to a lesser extent, in proximal convoluted tubules. The presence of different kinin-generating enzymes at these sites would permit the formation of kinins from appropriate substrates on both the vascular and luminal poles of separate segments of the kidney tubule.

Ultrastructural responses by Golgi apparatus of rat submandibular gland to β-adrenergic, α-adrenergic, and cholinergic stimulation

Abstract Rat submandibular gland tissue pieces were stimulated in vitro for 30 min with a β-adrenergic agent or a cyclic AMP analog to stimulate protein secretion, or with α-adrenergic or cholinergic agents or a Ca2+ ionophore to stimulate fluid secretion. Acinar cells were examined by transmission electron microscopy. In control tissue, acinar cells showed little evidence of secretory activity. The Golgi apparatus was sparse and was associated with a few small, immature secretory granules with fine fibrillar contents. Following secretory granule discharge stimulated by isoproterenol or dibutyryl cyclic AMP, acinar cells were constricted, and had extensive basolateral membrane folding and tightly packed rough endoplasmic reticulum. Golgi complexes were prominent and had multiple small granules with filamentous contents. After stimulation of fluid secretion by α-adrenergic agents (epinephrine, phenylephrine), or cholinergic agents (acetylcholine, carbachol, pilocarpine), or a Ca2+ ionophore (A23187), the Golgi apparatus had compact concave cisternae enclosing aggregates of tubulovesicles. Acinar cells were distended, basolateral membranes were expanded, and rough endoplasmic reticulum was dilated and vesiculated.