Kallikrein-like prorenin-converting enzymes in inbred hypertensive mice

Kallikreins are a group of serine proteases and are distinguished by having serine residue at their active site. Their general function is to convert inactive pro-peptide into its biologically active form. In recent years, emerging evidence indicates that some kallikrein-kinin enzymes also play a role in the modulation of renin-angiotensin system. These kallikrein-like prorenin converting enzymes act on renin-angiotensin by converting prorenin into biologically active renin. In this investigation, kallikrein-like prorenin converting enzyme (PRCE C) (mK9) is isolated from genetically inbred high blood pressure (BPH) and their normal counterparts (BPN) mice, and its protein levels are quantitated. Levels of mRNA expression are also compared. Additionally, localization of the enzyme is visualized by in situ hybridization histochemistry. Results indicated higher levels of PRCE C (mK9) enzyme in BPH mice in comparison to their normal counterparts. mRNA expression was also higher in BPH mice. In situ hybridization histochemistry results localized PRCE C (mK9) in the striated duct cells of submandibular gland.     

Relationship between expression of epidermal growth factor and simian virus 40 T antigen in a line of transgenic mice

The pattern of expression of the simian virus 40 (SV40) T antigen gene and resultant dysplasia were re-examined in a line of transgenic mice in which the T antigen gene was under the control of the SV40 early promoter. We found that T antigen expression in the kidney, and resulting dysplastic lesions, occurred exclusively in the distal convoluted tubules and the ascending limbs of Henle. Epidermal growth factor (EGF) expression in the kidney of normal mice was similarly immunolocalized. The correlation between high EGF immunoreactivity in normal mouse tissues and T antigen expression in the transgenic counterpart was also seen in the choroid plexus epithelium and in the submandibular glands of male mice. T antigen was not found in the submandibular gland of transgenic females. Similarly, EGF was only rarely detected in the normal female submandibular gland. In contrast to the correlation between T antigen expression in the transgenic mice and EGF expression in the corresponding tissues of the normal mice, within the dysplastic lesions of the transgenic mice EGF expression was severely diminished. Adenocarcinomas of the male submandibular gland from another line of transgenic mice that expresses theInt-1 transgene, showed similarly reduced levels of immunostaining for EGF. Thus, reduced expression of EGF might be a general feature of dysplasia and tumorigenesis in those tissues that normally express EGF.     

Nerve growth factor-like immunoreactivities in rodent salivary glands and testis

Summary A series of polyclonal affinity-purified antibodies against mouse submandibular-gland nerve growth factor (NGF) are described. Using the submandibular gland of the male mouse and indirect immunofluorescence, the specificity and sensitivity of affinity-purified immunoglobulins and various other fractions from the immunized animals have been tested. It will be shown that affinity-purification schemes, including pre-purification of protein A-fractionated immunoglobulins to remove antibodies that bind to unrelated hydrophilic and hydrophobic proteins, significantly enhance the signal-to-noise ratio and specificity of the antibodies. The antibodies effectively detect NGF-like immunoreactivity in both fresh and fixed glandular tissue. Optimal fixation procedures are described. Fluorescence intensities are linearly correlated to log antibody concentration. By use of the best antibody fractions and optimal fixation protocols, the distribution of NGF-like immunoreactivity is described in eight different salivary glands (rat and mouse, male and female, submandibular and sublingual glands). In addition to the well-known large numbers of immunoreactive cells in the submandibular gland of the male mouse, immunoreactive cells were found in the sublingual gland of male mice and in the submandibular and sublingual glands of female mice. One antibody revealed a weak specific fluorescence also in the submandibular gland of the male mouse. In a survey of genital organs of male mice, one antibody revealed fluorescence in the germ cell line. We conclude that several polyclonal affinity-purified antibodies have been characterized that show a strong NGF-dependent binding to the secretory granules of tubular cells in the submandibular gland of male mice. These antibodies should make it possible to locate endogenous and perturbed NGF levels immunocytochemically, e.g., in the peripheral and central nervous system, where NGF concentrations may be several orders of magnitude lower than in the salivary glands.     

Blood flow of the right and left submandibular gland during unilateral carotid artery occlusion in rat: role of nitric oxide

The aim of the present study was to investigate the effect of unilateral carotid artery occlusion on the blood flow of submandibular gland in anesthetized rats and identify the role of nitric oxide (NO) in blood flow changes after the artery occlusion. L-NAME (N omega-nitro-L-arginine-methyl-ester; 10 mg/kg/day, per os) dissolved in tap water was used to block nitric oxide synthase. Glandular blood flow was measured using Sapirstein's indicator (86Rb) distribution technique. In the control animals the blood flow of left (ligated side) submandibular gland was lower than in the right (unligated side) one (right: 76.4+/-15.4 ml/min/100 g, 64.1+/-13.4 ml/min/100 g, p<0.01). The blood flow of submandibular glands decreased in NOS blocked group versus control. The vascular resistance after L-NAME treatment was elevated (control: 11+/-2.3 R/kg, L-NAME: 17.5+/-4.1 R/kg, p<0.001). In L-NAME group the difference between blood flow value of the left and right submandibular gland was significantly lower than in the control group (control: -16%, NAME: -8%, p<0.01). Conclusion: The maintenance of the blood flow in the left submandibular gland during ligation of the left common carotid artery could be due to the good vascular anastomotic system at these regions and adaptation of the submandibular vessels to the decreased perfusion pressure. Nitric oxide may have a role in the regulation of blood flow tinder this condition.     

Ablation of renin-expressing juxtaglomerular cells results in a distinct kidney phenotype

Renin-expressing cells are peculiar in that they act as differentiated cells, producing the hormone renin, while they also seem to act as progenitors for other renal cell types. As such, they may have functions independent of their ability to generate renin/angiotensin. To test this hypothesis, we ablated renin-expressing cells during development by placing diphtheria toxin A chain (DTA) under control of the Ren1d mouse renin promoter by homologous recombination in a two-renin gene strain (Ren2 and Ren1d). Renin-expressing cells are essentially absent from kidneys in homozygotes (DTA/DTA) which, unlike wild-type mice, are unable to recruit renin-expressing cells when homeostasis is threatened. In contrast, renin staining in the submandibular gland (SMG), which expresses mainly Ren2, is normal. Homozygous mice survive normally, but the kidneys are small and have morphological abnormalities: 25% of the glomeruli are hyperplastic or atrophic, tubules are dilated and atrophic, and areas of undifferentiated cells exist near the atrophic glomeruli and tubules. However, in contrast to the very abnormal renal vessels found when renin-angiotensin system genes are deleted, the kidney vessels in homozygotes have normal wall thickness and no decrease in lumen size. Homozygotes have severely reduced kidney and plasma renin concentrations and females have reduced blood pressure. Homozygotes have elevated blood urea nitrogen and potassium levels, which are suggestive of altered renal function. We conclude that renin cells per se are necessary for the morphological integrity of the kidney and may have a role in maintenance of normal kidney function.     

Isolation and characterization of a protein corresponding to mKlk-11 clone from male mouse submandibular gland

A protein corresponding to the predicted genomic sequence of clone mKlk-11 has been characterized from mouse submandibular gland. This protein was purified by reverse-phase high-performance liquid chromatography from the submandibular glands of normal and hypertensive mice. The protein was not detected in the submandibular gland of mice selected for low blood pressure. It consists of three fragments starting at the residues 1, 98, and 141 of the predicted sequence of clone mKlk-11. The cleavage of-lactoglobulin (between residues 20 and 21, Tyr-Ser, and 40 and 41, Arg-Val) and a synthetic renin substrate tetradecapeptide (residues 4 and 5, Tyr-Ile) by the protein corresponding to clone mKlk-11 showed both tryptic- and chymotryptic-type cleavages. The possibility of this protein's involvement in the regulation of local blood flow is raised.     

Renin substrate and the renin-angiotensin system in hog tissues

The individual components of the renin-angiotensin system has been identified in numerous tissues. In this study we have examined whether a functional renin-angiotensin system is operative in several hog tissues including brain, aorta, and liver. The contribution of tissue renin substrate to the rate of local angiotensin generation was also assessed. Electrophoretic differences in plasma and tissue renin substrates, indicating structural differences, were employed as an index of independence of the tissue system from that of the peripheral circulation. Our results indicate that all tissues studied had the potential to locally generate angiotensin and that renin substrate limited to rate of the renin reaction in these tissues. Electrophoretic parameters, polyacrylamide gel electrophoresis, and isoelectric focusing suggest that the tissue renin systems are of local origin. The potential magnitude of local angiotensin production is such that tissue renin-angiotensin systems may significantly contribute to the control and regulation of blood pressure and other regulatory mechanisms influenced by angiotensin.     

Involvement of angiotensin II and endothelin-1 in the development of submandibular gland hypertrophy in response to isoproterenol in rats

We investigated whether angiotensin II (Ang II) and endothelin-1(ET-1) are involved in submandibular hypertrophy in response to repeated treatment with isoproterenol (ISO) in rats. The immunoreactive Ang II (IRAng II) and immunoreactive ET-1 (IRET-1) contents of ISO-induced hypertrophy were significantly higher than those of control glands. Treatment of isolated gland tissues with ISO (1 microM) or dobutamine (1 microM) caused significant increases in the IRAng II and IRET- 1 contents of the glands compared with controls. These increases were suppressed by pretreatment with enalapril (3 microM) or captopril (3 microM). Treatment with Ang II (10 microM) also caused an increase in IRET-1 content. Our findings suggest that Ang II and ET-1 are involved in the submandibular gland hypertrophy that develops in rats repeatedly treated with ISO, and that these biologically active peptides may act as growth factors. They also imply that the tissue renin-angiotensin system and Ang II specific receptors are present in the submandibular glands.     

A pituitary-salivary mixed gland induced by tissue recombination of embryonic pituitary epithelium and embryonic submandibular gland mesenchyme in mice

Renal subcapsular syngrafts of Day 9 to 11 mouse embryonic pituitary epithelium with Day 14 mouse embryonic submandibular gland mesenchyme produced mixed organs that include residual cleft structure surrounded by anterior pituitary cells some which are stained by anti-ACTH antiserum and submandibular gland-like structure with differentiated acinar cells which are stained by anti-alpha-amylase antiserum. However, when Day 8.5 or 12 embryonic pituitary epithelium was recombined with submandibular gland mesenchyme and syngrafted, development of submandibular gland-like or anterior pituitary tissues resulted, respectively. Thus, during organogenesis of the mouse anterior pituitary, there exists a developmental stage (Day 8.5-11 in utero), when prospective pituitary epithelium can respond to heterotypic submandibular gland mesenchyme with the development of a submandibular gland-like tissue.     

Role of the renal nerve in glucocorticoid hypertension

The present study was designed to investigate the involvement of the renal nerve in glucocorticoid hypertension and to assess the role of the renin-angiotensin system in dexamethasone-induced hypertension. The elevated blood pressure in dexamethasone treated rats showing a significant increase in plasma renin concentration (PRC) and activity (PRA) was attenuated dose-dependently by the angiotensin I converting enzyme (ACE) inhibition. Bilateral renal denervation caused a partial decrease in the elevated blood pressure, abolished the increased PRC and PRA, and reduced the dose-dependent decrease in blood pressure with ACE inhibition in dexamethasone treated rats. Although the reduction in body weight and increases in urine volume, urinary sodium excretion and hematocrit were clearly seen following dexamethasone administration, dexamethasone-treated renal denervated rats showed the same degree of change in any of the variables as dexamethasone-treated sham-operated rats. Thus, our results indicate that the stimulation of the renin-angiotensin system through the activation of the renal nerve may be partially responsible for the dexamethasone-induced high blood pressure and, therefore, bilateral renal denervation reduces, partially but significantly, the elevated blood pressure, suggesting that the attenuation of oversecretion of renin contributes to the lowering of the blood pressure.     

Angiotensin II and aldosterone regulation

The circulating renin-angiotensin system is a major regulator of the secretion of the adrenocortical hormone, aldosterone. This renin-angiotensin aldosterone system is important in the control of salt and water balance and blood pressure. This review describes the historical background leading to the discovery of aldosterone in the 1950s and the recognition in the 1960s that angiotensin II was involved in its control. Although angiotensin II is important in the regulation of aldosterone secretion, its action is influenced by multiple other factors, especially potassium and atrial natriuretic peptide. In addition to the circulating renin-angiotensin system, a local renin-angiotensin system is present in the zona glomerulosa cell. This local system also appears to be involved in the regulation of aldosterone production. The mechanism by which angiotensin II stimulates the adrenal zona glomerulosa cell is described in some detail. Angiotensin II interacts with the angiotensin receptor (AT1) membrane receptor that is coupled to cellular second messengers. Specific AT1 receptor antagonists are now clinically used to block angiotensin II's action on various target organs, including the adrenal gland.     

C-Fos在db/db自发性糖尿病小鼠颌下腺的表达

目的观察转基因糖尿病小鼠下颌下腺形态学改变与原癌基因C-fos蛋白表达的关系,为糖尿病的临床及基础研究提供依据。方法引进日本C57BL/ksj-db/ m表型正常隐性基因小鼠,其近亲交配所得纯合子后代,即为db/db(单基因遗传自然发病型)糖尿病小鼠。取3、4、6、8、10月龄db/db糖尿病小鼠及相应月龄的db/ m正常小鼠下颌下腺,行HE染色及SP免疫组化染色后进行图象分析,统计各组下颌下腺C-fos阳性表达的细胞数,观察其形态学改变。结果糖尿病小鼠下颌下腺腺泡萎缩,细胞缩小,形态不规则,排列不整齐。各月龄糖尿病小鼠颌下腺C-Fos阳性细胞明显低于相应对照组(P<0.01),且逐渐减少,呈下降趋势。结论db/db糖尿病状态下颌下腺细胞表达C-Fos蛋白明显降低,c-fos低表达可能与下颌下腺实质细胞的增殖减弱性形态学变化密切相关。     

Intracellular signalling cascades activated by the EGF receptor and/or by integrins, with potential relevance for branching morphogenesis of the fetal mouse submandibular gland

The fetal mouse submandibular gland grown in vitro is a valuable model system to study epithelio-mesenchymal interactions vital for branching morphogenesis. This basic developmental process is dependent upon simultaneous engagement of receptors for growth factors and components of the extracellular matrix. Here we review intracellular signalling cascades mediated by activation of the EGFR in the developing mouse submandibular gland.     

小鼠颌下腺降钙素基因相关肽的免疫组织化学定位

本文用免疫组织化学ＡＢＣ法,对小鼠颌下腺中降钙素基因相关肽（ＣａｌｃｉｔｏｎｉｃＧｅｎｅ－ＲｅｌａｔｅｄＰｅｐｔｉｄｅ,ＣＧＲＰ）的分布进行了观察。结果表明：小鼠颌小腔内有降钙素基因相关肽免疫反应神经的分布,它们主要分布于小叶间结缔组织中。此外,颗粒由管细胞也呈降钙素基因相关肽免疫反应阳性。提示降钙素基因相关肽可能参与颌下腺的分泌活动及血液循环等生理调节。     

Tissue distribution and characterization of prorenin-converting enzyme in mouse

Renin is produced from an inactive precursor, prorenin, through endoproteolytic cleavage at paired basic amino-acid residues. Using (35S)methionine-labeled prorenin, that was synthesized with Xenopus oocyte expression system, as a substrate, we have determined the tissue distribution and the nature of prorenin-converting activity in mouse. The highest activity was found in the submandibular gland of male ICR mouse. The activity of the enzyme seemed to be parallel to that of renin. This enzyme activity, with an optimal pH 8.0-8.5, was inhibited by leupeptin, antipain and benzamidine.     

Reduced levels of insulin-like growth factor-1 receptor (IGF-1R) suppress cellular signaling in experimental autoimmune sialadenitis (EAS).

The nonobese diabetic mouse (NOD) develops destruction and functional impairment of salivary and lachrymal glands, experimental autoimmune sialadenitis (EAS), resembling and representing a model for Sjogren's syndrome (SS). To investigate the mechanisms of tissue destruction in EAS, we analyzed a cell survival promoter insulin-like growth factor-1 receptor (IGF-1R) in the submandibular glands of NOD mice with this disease. We also evaluated the expression of a downstream effector of IGF-1R, BAD. Receptor-binding autoradiography revealed that the IGF-1R levels in submandibular glands from young NOD mice were lower than those in adult NOD mice. Immunofluorescence staining demonstrated that BAD expression in the epithelial cells of the submandibular gland was consistently enhanced throughout the course of EAS in NOD mice. These findings suggest that a reduction in the levels of IGF-1R induces a defective glandular homeostasis in the submandibular gland epithelial cells and triggers EAS.     

Defective epidermal growth factor gene expression in mice with polycystic kidney disease

The C57BL/6J-cpk mouse has an inheritable form of polycystic kidney disease similar to the autosomal recessive disorder seen in humans. Between approximately 1 and 3 weeks of age, affected cpk mice develop numerous large cysts in the collecting tubule segment of kidney nephrons. The present study examined the ontogeny of renal and submandibular gland prepro-epidermal growth factor (preproEGF) gene expression in the cpk mouse using Northern blot hybridization and immunohistochemistry. There was a virtual absence of renal preproEGF gene expression in cystic kidneys over the 3-week postnatal period, during which time renal preproEGF mRNA and proEGF/EGF protein normally reach significant levels. PreproEGF mRNA was expressed in salivary glands of cystic mice; however, this mRNA could not be further elevated with testosterone suggesting that there are abnormalities in the regulation of the preproEGF gene in the submandibular gland, as well as in the kidney. Since renal preproEGF expression during the early postnatal period occurs when collecting duct cysts form, it is possible that a deficiency in renal proEGF or EGF contributes to the rapid development of collecting duct cysts and the concomitant renal failure in the C57BL/6J-cpk cystic mouse.     

Localization of a mouse submandibular sialomucin by indirect immunofluorescence

Summary A sialomucin from the mouse submandibular gland was localized in the gland by indirect immunofluorescence. Fluorescence was localized over the acinar cells and, to a lesser extent, in the lumen of the ducts. The mucin antiserum did not show cross-reactivity with cells from the sublingual gland or with other mucous-producing cells from the respiratory and gastrointestinal tracts of the mouse, or with salivary gland tissue of the rat. The sialomucin lacks both sulphate andl-fucose. Localization of a mucin with such a composition, within acinar cells of the mouse submandibular gland, is consistent with previous observations from histochemistry and autoradiography.     

体外培养小鼠下颌下腺细胞生长特性的实验研究

目的：研究小鼠下颌下腺细胞的培养方法,探讨下颌下腺细胞培养条件及细胞生长特性,为研究干细胞转分化涎腺腺泡细胞以及涎腺再生研究奠定理论基础和技术支持。方法：取2周龄的小鼠,组织块法和消化法分别进行培养,用活细胞观察法和HE染色法记录细胞形态学特征;免疫荧光染色法鉴定;通过生长曲线和细胞倍增时间来比较两种方法对细胞增殖能力的影响。结果：组织块法和消化法均可以成功的培养下颌下腺细胞,（1）组织块法培养的细胞呈卵圆形或多边形,10天左右成铺路石样;消化法培养细胞亦为上皮样细胞,呈多边形,胞质丰富;（2）HE染色下颌下腺细胞呈多边形,胞核明显;（3）Cytokeratin13和AQP5表达阳性,Wimentin表达阴性;（4）组织块法培养细胞增殖相对较平缓,消化法培养细胞增长较迅速;（5）消化法培养倍增时间（1．3471±0．6071）天比组织块法倍增时间（2．1887±1．1503）明显缩短（P〈0．05）;结论：体外可以成功的培养下颌下腺细胞,但是同时证明下颌下腺细胞长期传代较困难,这为研究干细胞转分化涎腺细胞和涎腺再生体内实验提供了理论和实验基础。