Caveolae, caveolin and cav-p60 in smooth muscle and renin-producing cells in the rat kidney

AIMS: In vascular smooth muscle cells caveolae are important for signalling mechanisms regulating vascular contraction. In smooth muscle layer of the renal afferent arteriole juxtaglomerular cells (JG cells) are non-contractile renin producing cells that have the capacity to change their phenotype into smooth muscle cells and back again by metaplastic transformation. Signalling mechanisms in JG cells are not fully understood and we therefore investigated if caveolae were present, and thereby could be involved as integrators of cellular signalling in both of these phenotypes of smooth muscle cells. METHODS: Using electron microscopy we compared the number of caveolae in JG cells and smooth muscle cells in the afferent arteriole of the rat kidney. The expression of caveolin and cav-p60 was examined using a combination of immunogold electron microscopy and immunofluorescence microscopy. RESULTS: We found that JG cells have sixfold less caveolae per cell surface sectional length than smooth muscle cells. The expression of cavolin-1 and cav-p60 correlated with the number of caveolae. An examination of the general distribution of caveolae, cav-p60 and caveolins in the rat kidney showed that cav-p60, like caveolin-1, is a specific maker of caveolae. CONCLUSION: The number of caveolae in JG cells is very low, and this makes it unlikely that caveolae are of major importance for the renin secretion specific for JG cells.     

Alveolar Epithelial Type II Cells and Their Microenvironment in the Caveolin‐1‐Deficient Mouse

Caveolin‐1 (Cav‐1) is highly expressed in alveolar epithelial type I (AE1) and endothelial cells of the alveolar region of the lung. Interestingly, alveolar epithelial type II (AE2) cells that are progenitors of the AE1 cells do not express Cav‐1. We investigated whether genetic Cav‐1 deficiency alters the phenotype of AE2 cells and their microenvironment using stereology. Total number, mean volume, and subcellular composition of the AE2 cells were not altered in Cav‐1 ^?/? when compared with wild‐type mice. The alveolar septa were thickened and contained a significantly greater volume of extracellular matrix. Thus, AE2 cells as progenitors of AE1 cells are not critically involved in the severe pulmonary phenotype in Cav‐1‐deficient mice. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.     

Expression of caveolin‐3 in the fibroblast‐like type B synoviocytes in the rat temporomandibular joint

The present study revealed that the fibroblast‐like type B synoviocytes (covering the surface of the synovial membrane in the rat temporomandibular joint) had muscle‐specific caveolin‐3 protein in their caveolae. The existence of two kinds of type B synoviocytes (with and without caveolin‐3‐immunoreactions even in the synovial lining layer) might reflect the functional difference between them. Anat Rec, 2007. © 2007 Wiley‐Liss, Inc.     

应用改良的免疫胶体金包埋方法检测p38蛋白激酶在Raw细胞中的定位

目的:采用两种包埋方法Lowicryl K4M低温包埋和改良Spurr包埋,观察p38在Raw264.7细胞中的免疫电镜定位,摸索出一种操作简单、适用于南方潮湿多雨天气的免疫电镜包埋方法。方法:分别采用低温包埋剂Lowicryl K4M和改良Spurr树脂包埋两种方法,经包埋后染色,观察p38蛋白激酶在Raw264.7中的超微结构分布。结果:改良Spurr树脂包埋后结果显示,未受刺激的及EGF刺激的Raw264.7中,p38在胞浆和胞核中金颗粒弥散在细胞的各个部分,受到LPS刺激后,细胞核区的金颗粒明显增多,而胞浆区域的金颗粒显著减少,观察到的Raw细胞超微结构清晰、免疫胶体金定位准确,与低温包埋剂Lowicryl K4M包埋结果完全一致。结论:相对于Lowicryl K4M,Spurr树脂价格低廉,粘度低、抗湿性能好、切割性能佳、操作简单,是一种适用于南方潮湿多雨天气的免疫电镜包埋剂,可以替代低温包埋剂Lowicryl K4M。     

Ultrastructural Immunolabeling in the Evaluation, Diagnosis, and Characterization of Neuroendocrine Neoplasms

Neuroendocrine neoplasia represents a heterogenous entity with variable morphologic light microscopic expressions. In many cases a definite diagnosis is easily made by light microscopic examination, but in some cases this does not suffice. In the latter instances, immunocytochemistry, ultrastructural examination, or both are required to diagnose a neuroendocrine neoplasm. However, basing a diagnosis of neuroendocrine neoplasia exclusively on the results obtained from immunocytochemical or ultrastructural evaluation of these tumors may not be entirely accurate in some instances. Ultrastructural immunolabeling plays a key role in accurately defining localization of immunoreactive substances in well-characterized neuroendocrine neoplasms, can assess colocalization of antigenic epitopes, helps define specificity and significance of immunocytochemistry results obtained at the light microscopic level, and is more sensitive than light microscopic immunocytochemistry. Some evolving diagnostic entities can be further characterized by utilization of ultrastructural labeling techniques. Controversies concerning the neuroendocrine nature of electron-dense structures identifiable at the ultrastructural level can be readily and accurately resolved. By providing a way to evaluate combined immunomorphologic parameters, ultrastructural immunogold labeling can settle important questions pertaining to neuroendocrine neoplasia. The present article illustrates a series of cases where the above-mentioned applications were tested.     

Detection of smooth muscle myosin in the theca cells of the ovaries

Smooth muscle myosin was detected by the indirect Coons' method, using an appropriate monospecific immune serum, in the theca cells of the ovaries in rodents.Laboratory of Age Pathology, Institute of Human Morphology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 80, No. 9, pp. 95–97, September, 1975.     

Melatonin release by exocytosis in the rat parotid gland

Several beneficial effects on oral health are ascribed to melatonin. Due to its lipophilic nature, non‐protein‐bound circulating melatonin is usually thought to enter the saliva by passive diffusion through salivary acinar gland cells. Recently, however, using transmission electron microscopy (TEM), melatonin was found in acinar secretory granules of human salivary glands. To test the hypothesis that granular located melatonin is actively discharged into the saliva by exocytosis, i.e. contrary to the general belief, the β‐adrenergic receptor agonist isoprenaline, which causes the degranulation of acinar parotid serous cells, was administered to anaesthetised rats. Sixty minutes after an intravenous bolus injection of isoprenaline (5 mg kg^?1), the right parotid gland was removed; pre‐administration, the left control gland had been removed. Samples were processed to demonstrate melatonin reactivity using the immunogold staining method. Morphometric assessment was made using TEM. Gold particles labelling melatonin appeared to be preferentially associated with secretory granules, occurring in their matrix and at membrane level but, notably, it was also associated with vesicles, mitochondria and nuclei. Twenty‐six per cent of the total granular population (per 100 μm² per cell area) displayed melatonin labelling in the matrix; three‐quarters of this fraction disappeared (P < 0.01) in response to isoprenaline, and melatonin reactivity appeared in dilated lumina. Thus, evidence is provided of an alternative route for melatonin to reach the gland lumen and the oral cavity by active release through exocytosis, a process which is under the influence of parasympathetic and sympathetic nervous activity and is the final event along the so‐called regulated secretory pathway. During its stay in granules, anti‐oxidant melatonin may protect their protein/peptide constituents from damage.     

Ultrastructure of the capillary pericytes and the expression of smooth muscle α‐actin and desmin in the snake infrared sensory organs

The infrared sensory membranes of pit organs of pit vipers have an extremely rich capillary vasculature that forms many vascular loops, each serving a small number of infrared nerve terminals. We clarified the ultrastructure of capillary pericytes in the pit membranes by scanning and transmission electron microscopy, and examined the immunoreactivity in their cytoplasm to two contractile proteins: smooth muscle α‐actin (SM α‐actin) and desmin. The capillary pericytes had two major cytoplasmic processes: thickened primary processes that radiate to embrace the endothelial tube and flattened secondary processes that are distributed widely on the endothelium. Coexpression of SM α‐actin and desmin was observed in the pericytes of entire capillary segments, and SM α‐actin was characterized by prominent filament bundles directed mainly at right angles to the capillary long axis. This expression pattern was different from that of capillary pericytes of the scales, where SM α‐actin was expressed diffusely in the cytoplasm. In a series of electron microscopic sections, we often observed the pericyte processes depressing the endothelial wall. We also observed a close relationship of the pericytes with inter‐endothelial cell junctions, and pericyte processes connected with the endothelial cells via gap junctions. From these findings, we surmised that capillary pericytes in the pit membrane have a close functional relationship with the endothelium, and through their contractile and relaxing activity regulate capillary bloodflow to stabilize production of infrared nerve impulses. Anat Rec 260:299–307, 2000. © 2000 Wiley‐Liss, Inc.     

Interactions between COX‐2 and the renin–angiotensin system in the kidney

Aim: In adult mammalian kidney, COX‐2 expression is found in a restricted subpopulation of cells. The two sites of renal COX‐2 localization detected in all species to date are the macula densa (MD) and associated cortical thick ascending limb cells (cTALH) and medullary interstitial cells. Physiological regulation of COX‐2 in these cellular compartments suggests functional roles for eicosanoid products of the enzyme. In the MD region, COX‐2 expression increases in high renin states [salt restriction, angiotensin converting enzyme (ACE) inhibition, renovascular hypertension], and selective COX‐2 inhibitors significantly decrease plasma renin levels and renal renin activity and mRNA expression. An important role for COX‐2‐derived prostanoids in regulation of renin expression and secretion has also been determined by using mice with selective genetic deletion of either the COX‐1 or COX‐2 gene. There is evidence for negative regulation of MD/cTALH COX‐2 by angiotensin II and by glucocorticoids and mineralocorticoids, suggesting that in the kidney, cortical COX‐2 expression is regulated in part by alterations in activity of the renin–angiotensin system.     

Morphologic changes in the renal glomerulus and the juxtaglomerular apparatus in human preeclampsia

The renal biopsies of ten women with preeclampsia without other underlying renal disease were examined in detail using light and electron microscopy and immunofluorescence. Characteristic preeclamptic glomerular lesions with endocapillary cell swelling, subendothelial and mesangial deposits, and mesangial interposition were detected in each patient. Juxtaglomerular regions were not prominent and were poorly granulated on light microscopy; ultra-structurally, they showed myoepithelioid cells with sparse renin granulation and considerable heterogeneity of granule size and density in association with relatively meagre granular endoplasmic reticulum and Golgi profiles. These morphologic findings suggest that, in patients with clinical and renal biopsy evidence of preeclampsia, there is no significant stimulation of the renin–angiotensin system.     

Capillary growth in overloaded,hypertrophic adult rat skeletal muscle: An ultrastructural study

We examined the early stages of angiogenesis in overloaded m. extensor digitorum longus following extirpation of the agonist m. tibialis anterior. Capillary-to-fibre ratio increased after 1 week (1.54 ± 0.02) vs. control (1.38 ± 0.06; P < 0.01) and resulted in a greater tortuosity of the capillary bed at 2 weeks, indicating the presence of lateral sprouts or anastomoses. Capillary endothelial cells (ECs) showed ultrastructural signs of activation, were thickened, and had irregular luminal and abluminal surfaces. The proportion of ECs with abluminal processes increased after overload (13.5 ± 0.6% vs. 2.0 ± 1.5%, 1 week vs. contralateral, P < 0.01; 12.5 ± 2.6% vs. 3.5 ± 0.6%, 2 weeks vs. contralateral, P < 0.01), whereas there was no significant change in proportion of luminal processes. Abluminal processes occurred in approximately 13% of capillaries in overloaded muscles (P < 0.01 v. control and contralateral), and most were associated with focal breakage of the basement membrane (BM). Small sprouts (<3 μm in diameter) comprised of one or two ECs sometimes lacked a lumen, and others had a slitlike or vacuolelike lumen between adjacent ECs or vacuolelike lumen formed by fusion of vesicles within a single EC. Endothelial mitosis was occasionally seen in nonsprouting capillaries with intact BM, increasing the average number of ECs per capillary from approximately 1.7 in control muscles to 2.1 after 1 week of overload (P < 0.05) when bromodeoxyuridine incorporation was also higher (P < 0.001). We conclude that muscle overload induces capillary growth by sprouting of existing capillaries, probably due to mechanical stretch acting from the abluminal side of the vessels. Anat. Rec. 252:49–63, 1998. © 1998 Wiley-Liss, Inc.     

Comparison of sarcomere alterations after muscle contraction and tension loading in the rat soleus muscle

Muscle contraction induced by 30 min of continuous nerve stimulation at 50 Hz resulted in sarcomere changes of the soleus muscle in the rat in our previous study. To further investigate the cause of sarcomere alterations, the sciatic nerve was electrically stimulated intermittently for 30 min. Nerve stimulation was also conducted after cutting the tendons of the soleus, gastrocnemius and plantaris muscles in order to prevent imposing tension on these muscles as a result to their own contractions. In addition, the muscles were pulled by weights via their tendons to load high tension for 30 min without nerve stimulation. Sarcomere alterations immediately after treatments were quantified by electron microscopy. The percentages of aberrant sarcomere areas of the soleus muscle were 25.7 ± 16.4% (mean ± SD) in the group of intermittent nerve stimulation with intact tendons and 21.1 ± 35.4% in the group of tenotomy and continuous nerve stimulation, which were roughly equal to or more severe than the group of continuous nerve stimulation with intact tendons (18.8 ± 15.8%) in our previous study. Sarcomere alterations consisted mainly of hypercontraction in these groups. Almost all sarcomere changes in the tension-loaded (pulled) soleus muscles were scarce myofilaments (1.7 ± 1.0% by 600 g; 4.5 ± 2.9% by 1200 g), and hypercontraction was not observed. These findings indicate that neither high tension nor a decrease of muscle blood flow during continuous contraction seems to be the primary cause of sarcomere alterations in the present study. There are probably other causes that produce aberrant sarcomeres.     

Ontogeny of beta-adrenergic receptors in pulmonary arterial smooth muscle, bronchial smooth muscle, and alveolar lining cells in the rat.

beta-Adrenergic receptors play an integral role in the modulation of cell function in the developing lung. In the rat, there are marked increases in beta receptor density in whole lung during postnatal maturation, but it is now known whether there are differential developmental changes in receptor density in specific cell types. Quantitative light microscopic autoradiography with [125I]iodocyanopindolol ([125I]ICYP) was used to determine maturational changes in beta-adrenergic receptor density in pulmonary arterial smooth muscle (ASM), bronchial smooth muscle (BSM), and alveolar lining cells (ALC) in rat lung during postnatal development (1 day to 6 mo). [125I]ICYP binding to whole lung sections revealed a single class of high-affinity receptors; agonist competitive binding studies suggested that the receptors are primarily of the beta 2 subtype. beta-Adrenergic receptor density in newborn (1 day) lung was lowest in ASM cells and was comparable in BSM cells and ALC. In contrast, in lungs from adult rats (3 mo), receptor density was similar in ASM versus BSM cells and was 2-fold greater in ALC. In addition, the maturational pattern of increasing receptor density differed in ASM compared with BSM and ALC. Receptor density in ASM increased 93% from 1 to 13 days, another 92% from 13 to 20 days, and was unchanged thereafter. In contrast, receptor density in BSM cells did not change from 1 to 13 days, but it increased 65% from 13 to 20 days, rose another 47% from 20 days to 3 mo, and increased an additional 24% from 3 to 6 mo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estrogen and aging affect synaptic distribution of phosphorylated LIM kinase (pLIMK) in CA1 region of female rat hippocampus

17beta-Estradiol (E) increases axospinous synapse density in the hippocampal CA1 region of young female rats, but not in aged rats. This may be linked to age-related alterations in signaling pathways activated by synaptic estrogen receptor alpha (ER-alpha) that potentially regulate spine formation, such as LIM-kinase (LIMK), an actin depolymerizing factor/cofilin kinase. We hypothesized that, as with ER-alpha, phospho-LIM-kinase (pLIMK) may be less abundant or responsive to E in CA1 synapses of aged female rats. To address this, cellular and subcellular distribution of pLIMK-immunoreactivity (IR) in CA1 was analyzed by light and electron microscopy in young and aged female rats that were ovariectomized and treated with either vehicle or E. pLIMK-IR was found primarily in perikarya within the pyramidal cell layer and dendritic shafts and spines in stratum radiatum (SR). While pLIMK-IR was occasionally present in terminals, post-embedding quantitative analysis of SR showed that pLIMK had a predominant post-synaptic localization and was preferentially localized within the postsynaptic density (PSD). The percentage of pLIMK-labeled synapses increased (30%) with E treatment (P<0.02) in young animals, and decreased (43%) with age (P<0.002) regardless of treatment. The pattern of distribution of pLIMK-IR within dendritic spines and synapses was unaffected by age or E treatment, with the exception of an E-induced increase in the non-synaptic core of spines in young females. These data suggest that age-related synaptic alterations similar to those seen with ER-alpha occur with signaling molecules such as pLIMK, and support the hypothesis that age-related failure of E treatment to increase synapse number in CA1 may be due to changes in the molecular profile of axospinous synapses with respect to signaling pathways linked to formation of additional spines and synapses in response to E.     

Comparative studies of the same adenocarcinoma cells,macrophages, and mesothelial cells by light microscopy,scanning electron microscopy,and transmission electron microscopy

The identification of cells in body cavities of cancer patients is sometimes difficult to make. In order to make a definite cytological diagnosis, we observed the same cells by using light microscopy (LM)-scanning electron microscopy (SEM)-transmission electron microscopy (TEM). In this study we first stained cells by the Papanicolaou method after fixation in 1% glutaraldehyde for LM, and then attempted to observe them successively by SEM-TEM after fixation in 1% paraformaldehyde and 1.25% O^s0⁴. Our method and procedures in examining successively one and the same cells in body cavity fluids by using LM, SEM, and TEM ensured accurate discrimination among adenocarcinoma cells, mesothelial cells, and macrophages. The results of this study suggest that LM-SEM-TEM may be of diagnostic value in distinguishing among mesothelial cells, macrophages, and adenocarcinoma cells. This method also succeeded in disclosing differences between the ultrastructure of the cell surfaces, and those of the cytoplasm, and of the nuclei It is desirable that LM-SEM-TEM observation can be introduced into various aspects in order to obtain an improvement in the diagnosis by cytologic examination, the judgment of therapeutical effects, drug selection, and prognostic presumption. Diagn Cytopathol 1994; 11:333–342. © 1994 Wiley-Liss, Inc.     

PP60c-Src在血管紧张素II诱导的大鼠血管平滑肌细胞信息转导中的作用(英)

目的：本研究通过观察c-Src在AngII对大鼠血管平滑肌细胞(VSMC)丝裂原激活的蛋白激酶(MAPK)的激活和c-Fos蛋白表达中的影响，以进一步阐明AngII促VSMC增殖的细胞内信息转导机制。方法： 原代和传代培养SD大鼠主动脉VSMC，以脂质体包裹反义c-Src寡脱氧核苷酸(oligodeoxynucleotides, ODNs)转染培养的VSMC以抑制c-Src蛋白表达和激酶活性。以未转染的VSMC为对照，观察10-7mol/L AngII刺激对转染的VSMC的MAPK活性和c-Fos蛋白表达的影响。蛋白免疫沉淀和酶自身磷酸化率法测定c-Src激酶活性；髓鞘碱性蛋白(MBP)底物磷酸化率测定MAPK激酶活性；Western blotting免疫印迹法测定c-Src和c-Fos蛋白表达情况。结果： 转染不同浓度反义c-Src ODNs的VSMC，c-Src蛋白含量呈浓度依赖性降低，c-Src激酶活性也显著 抑制，以Ang II刺激经转染反义c-Src ODNs的VSMC，c-Src激酶活性增幅仅为对照组的8.7%；MAPK活性仅为对照的1.6%；c-Fos蛋白表达的增幅为对照组的30.0%。结论： AngII可诱导VSMC c-Src激活和细胞内信息转导，且AngII引起的MAPK和c-fos的激活依赖于c-Src的激活，提示c-Src是AngII促血管平滑肌细胞增殖的重要信息分子。     

Effects of hypovolaemia on acid‐induced duodenal mucosal damage in the rat

Modest acute hypovolaemia in rats markedly decreases the duodenal mucosal alkaline secretion via neurohumoral links. The present study was undertaken to investigate if such a procedure influences the morphological changes observed following an acid challenge of the duodenal mucosa. Experiments were performed on anaesthetized male Sprague–Dawley rats. HCl (10 or 100 mM ) was infused during 15 min into the duodenum via a luminally situated catheter. Time controls were compared with animals bled 10% of total blood volume. Mucosal damage was evaluated by light microscopic morphometry on transverse sections and by scanning electron microscopy of the luminal surface. Perfusion with either 10 mM or 100 mM HCl reduced villus length by about 30%. The villus area was unaffected by 10 mM HCl, but was reduced significantly by 10% by 100 mM HCl as compared with NaCl time controls. Hypovolaemia did not influence the morphometrical changes induced by 10 mM HCl but reduced significantly both villus length (–28%) and villus area (–10%) as compared with the unbled 100 mM HCl group. Scanning electrone microscopy (SEM)‐based visual damage score was not influenced by the hypovolaemia procedure in any of the acidities. Morphological changes of the duodenal mucosa, induced by moderate intra‐luminal acidity (10 mM HCl), is not influenced by hypovolaemia. However, at higher acidities (100 mM HCl) the hypovolaemia contributes to more severe mucosal damage.     

Nail-Patella syndrome in a spontaneously aborted 18-week fetus: Ultrastructural and immunofluorescent study of the kidneys

Nail-patella syndrome (NPS), hereditary onycho-osteodysplasia, is an autosomal dominant disorder of nail dystrophy, patellar absence or hypoplasia, incomplete elbow extension, conical posterior iliac horns, and nephropathy. We studied the kidneys of an 18-week spontaneously aborted fetus of a mother with the NPS. Ultrastructural examination of the kidney showed thickening of the capillary walls of the glomeruli and mesangium. There was irregular thickening of basement membranes with subendothelial fibrillar electron-dense deposits. Immunofluorescence showed fibrinogen deposition in glomerular basement membranes. Fibrinogen deposition in utero may ultimately lead to glomerular fibrosis and intrabasement membrane collagen deposition as seen in the adult renal lesion of this syndrome. This is the first report of the NPS in which the renal abnormalities have been studied in a fetus. These findings provide support for possible prenatal diagnosis of NPS by intrauterine kidney biopsy. © 1992 Wiley-Liss, Inc.     

The importance of smooth muscle cells in the development of foam cells in the gastric mucosa

Summary Foam cells in lipid islands of the stomach can develop from both histiocytes and smooth muscle cells. With increasing storage of lipid vacuoles in smooth muscle cells, loosening of the myofilament arrangement and decrease of the dense areas subjacent to the plasma membrane occurs. Endoplasmic reticulum and the cisternae of the Golgi-apparatus dilate, the cell organelles increase initially and the basement membrane of the smooth muscle cells is fragmentarily formed. Only in incompletely formed foam cells can the origin from smooth muscle cells be recognised, in their final state their histiogenesis is seldom apparent.The authors are grateful to H. Gerdes for leaving the specimens and to R. Naumann and H. Staubitz for technical and photographic assistance.