Structural-functional characteristics of the renal endocrine system after alpha-1 adrenoreceptor blockade]

Research Institute of Cardiology, Uzbek Health Ministry Structural and functional peculiarities of the renal juxtaglomerular and interstitial cells were studied after alpha 1-adreno-blockers were used. The endocrine cells were studied after a single and prolonged introduction of the agents. Administration of a single dose of the drugs led to hypergranulation in juxtaglomerular cells thus decreasing their functional activity and release of the granules from the cells. The interstitial cells function intensified. Prolonged administration of alpha 1-adrenoblockers caused a degranulation process in the juxtaglomerular cells and a decrease in their protein-synthetizing structures, namely, renin synthesis, and elevation of synthetic process in the interstitial cells.     

Work in progress correlation of renal medullary prostaglandin content and renal interstitial cell lipid droplets

Indomethacin administration and hydronephrosis in rabbits has been found to produce increases in the number and changes in the composition of the lipid droplets in the renal medullary interstitial cells. The response to indomethacin, a prostaglandin synthetase inhibitor, was dose dependent.Work is in progress to assess the effects of other non-steroidal antiinflammatory drugs on the renal inner medulla and the interstitial cell lipid droplets.     

Mast cell products stimulate collagenase and prostaglandin E production by cultures of adherent rheumatoid synovial cells

Mast cells were purified from histologically-confirmed dog mastocytomas and extracted for whole mast cell products (MCP). When added to cultures of human adherent rheumatoid synovial cells MCP induced a 50-400 fold increase in prostaglandin E synthesis and a 10-50 fold stimulation of collagenase production. The mast cell stimulatory factor has not been identified and was not due to histamine, heparin or prostaglandin E. These results indicate a novel way in which mast cells might interact with synovial cells to promote the production of inflammatory mediators and proteolytic enzymes which might contribute to connective tissue degradation.     

Developmental regulation of interstitial cell density in bullfrog skeletal muscle

Denervation of skeletal muscle results in striking connective tissue remodelling in junctional areas of muscle. Since extracellular matrix molecules mediate axonal growth and synaptic differentiation, it is likely that the interstitial cells and matrix molecules that accumulate near synaptic sites after denervation influence the regrowth and regeneration of synaptic connections. The experiments presented here addressed the question of whether the junctional connective tissue in developing bullfrog skeletal muscle was also specialized in its cellular and molecular composition. Denervation responses of muscle, such as extrajunctional sensitivity to acetylcholine, often reproduce the characteristics of developing muscle during synaptogenesis. In developing muscle, the distribution of interstitial cells was nonuniform during the period of muscle fibre birth and synaptogenesis. Interstitial cells were concentrated near synaptic sites as in denervated adult muscle. Unlike denervated adult muscle, there were no junctional accumulations of fibronectin or tenascin, matrix molecules produced by interstitial cells, in developing muscles. These results demonstrate that the junctional connective tissue in developing muscle is identified by a high density of interstitial cells that may play a role in the identification and formation of synaptic sites. Further, the junctional matrix environment of developing muscle is distinct from the matrix remodelling that occurs in response to denervation, suggesting that the matrix production by interstitial cells during development is regulated differently from that after denervation of the neuromuscular junction.     

Network organization of interstitial connective tissue cells in the human endolymphatic duct.

The human endolymphatic duct (ED) and sac of the inner ear have been suggested to control endolymph volume and pressure. However, the physiological mechanisms for these processes remain obscure. We investigated the organization of the periductal interstitial connective tissue cells and extracellular matrix (ECM) in four freshly fixed human EDs by transmission electron microscopy and by immunohistochemistry. The unique surgical material allowed a greatly improved structural and epitopic preservation of tissue. Periductal connective tissue cells formed frequent intercellular contacts and focally occurring electron-dense contacts to ECM structures, creating a complex tissue network. The connective tissue cells also formed contacts with the basal lamina of the ED epithelium and the bone matrix, connecting the ED with the surrounding bone of the vestibular aqueduct. The interstitial connective tissue cells were non-endothelial and non-smooth muscle fibroblastoid cells. We suggest that the ED tissue network forms a functional mechanical entity that takes part in the control of inner ear fluid pressure and endolymph resorption.     

Tumor necrosis factor-alpha stimulates the biosynthesis of matrix metalloproteinases and plasminogen activator in cultured human chorionic cells.

To investigate the role of tumor necrosis factor-alpha (TNF alpha) in advanced collagenolysis and degradation of connective tissue components in preterm parturition, the effects of human recombinant TNF alpha (hrTNF alpha) on the production of matrix metalloproteinase 1 (MMP-1)/tissue collagenase, MMP-3/stromelysin, tissue inhibitor of metalloproteinases (TIMP), urokinase type-plasminogen activator (uPa) and prostaglandin (PG) E2 in human chorionic cells were examined in vitro. Human chorionic cells, but not amniotic cells, were found to respond to macrophage-conditioned medium (contains mainly interleukin 1) to produce MMP-1 and MMP-3. This indicated that the chorionic cell is one of the MMP-producing cells of fetal membranes. When confluent chorionic cells were treated with hrTNF alpha, the production of MMP-1 and MMP-3 as well as of uPa and PGE2 was greatly increased in a dose-dependent manner. In contrast, the production of TIMP was suppressed by hrTNF alpha. These results suggested that TNF alpha may participate in destruction of collagen and other connective tissue matrix components of fetal membranes and in promotion of uterine contractility in preterm parturition with intraamniotic infection.     

Stimulation of prostaglandin synthesis in cultured rat synovial cells by a factor derived from polymorphonuclear leukocytes

Stimulation of synovial cell prostaglandin production by a factor obtained from casein-induced peritoneal polymorphonuclear (PMN) cells has been investigated. Both the extract and short time cultured medium of rat peritoneal PMN cells stimulate prostaglandin (PG)E2 production as well as collagenase production in the culture of rat synovial cells. PGE2 production by the cells in the presence of the PMN factor is much faster (5 to 24 hr) than collagenase production (24 hr or later, Biomedical Res. 3, 506-516, 1982). This stimulating factor is confirmed to be derived from PMN cells, based on the purification of the cells from peritoneal exudate cells by the Ficoll-Urographin method. Elution profile of the factor on gel filtration has indicated that both PGE2 and collagenase productions by synovial cells are stimulated by the same effluent fractions corresponding to molecular weights of 15,000 - 20,000 daltons and 30,000 - 40,000 daltons. These results suggest that PMN cells are involved in PG production as well as collagenase production in the inflamed tissue by stimulating connective tissue cells such as synovial cells.     

Interleukin 4 inhibition of prostaglandin E2 synthesis blocks interstitial collagenase and 92-kDa type IV collagenase/gelatinase production by human monocytes.

Activation of human monocytes results in the production of interstitial collagenase through a prostaglandin E2 (PGE2)-cAMP-dependent pathway. Inasmuch as interleukin 4 (IL-4) has been shown to inhibit PGE2 synthesis by monocytes, we examined the effect of IL-4 on the production of human monocyte interstitial collagenase. Additionally, we also assessed the effect of IL-4 on the production of 92-kDa type IV collagenase/gelatinase and tissue inhibitor of metalloproteinase-1 (TIMP-1) by monocytes. The inhibition of PGE2 synthesis by IL-4 resulted in decreased interstitial collagenase protein and activity that could be restored by exogenous PGE2 or dibutyryl cyclic AMP (Bt2cAMP). IL-4 also suppressed ConA-stimulated 92-kDa type IV collagenase/gelatinase protein and zymogram enzyme activity that could be reversed by exogenous PGE2 or Bt2cAMP. Moreover, indomethacin suppressed the ConA-induced production of 92-kDa type IV collagenase/gelatinase. These data demonstrate that, like monocyte interstitial collagenase, the conA-inducible monocyte 92-kDa type IV collagenase/gelatinase is regulated through a PGE2-mediated cAMP-dependent pathway. In contrast to ConA stimulation, unstimulated monocytes released low levels of 92-kDa type IV collagenase/gelatinase that were not affected by IL-4, PGE2, or Bt2cAMP, indicating that basal production of this enzyme is PGE2-cAMP independent. IL-4 inhibition of both collagenases was not a result of increased TIMP expression since Western analysis of 28.5-kDa TIMP-1 revealed that IL-4 did not alter the increased TIMP-1 protein in response to ConA. These data indicate that IL-4 may function in natural host regulation of connective tissue damage by monocytes.     

Effect of cadmium chloride on the ovulation and structure of ovary in the inbred KP and CBA mice strains

A single dose of cadmium chloride (2.2 mumol/100 g of body weight) brought about in the sensitive KP strain alterations of the ovarian structure and disturbances in the ovarian cycle manifested by a prolongation of diestrus. Following cadmium administration and increase in the amount of atretic follicles, especially those showing stages 1I and 2II of degeneration, was observed in the ovaries of KP mice. The corpora lutea contained numerous degenerated cells and were infiltrated by abundant connective tissue cells. The used dose of cadmium chloride showed no influences upon progesterone level. The absence of changes in the ovarian morphology and in the duration of the oestrus cycle in CBA females suggest that this strain is resistant to cadmium chloride.     

Endogenous steroid production in cellular and subcellular fractions of rat testis after prolonged treatment with gonadotropins.

Steroid production and enzyme activities were examined in preparations of whole testis tissue, isolated interstitial tissue and seminiferous tubules obtained from adult rats with intact pituitaries receiving daily subcutaneous injections of 100 I.U. human chorionic gonadotropin for 5 days and from control animals. After human chorionic gonadotropin administration testosterone concentrations were increased in total homogenates of whole testis tissue, interstitial tissue and seminiferous tubules. The testosterone production from endogenous precursors was enhanced only in total homogenates of whole testis tissue and interstitial tissue obtained from testes of human chorionic gonadotropin-treated rats. The production of testosterone in the corresponding homogenates of isolated seminiferous tubules was very low. The specific activity of 3 beta-hydroxysteroid dehydrogenase was increased in total homogenates of whole testis tissue, isolated interstitial tissue and seminiferous tubules. No effect was observed on the specific activities of marker enzymes such as cytochrome c oxidase, monoamine oxidase, steroid sulfatase and lactate dehydrogenase, whereas the specific activities of carboxyl esterase were decreased in homogenates of whole testis tissue and interstitial tissue. Total activity of monoamine oxidase was increased in homogenates of interstitial tissue of tests from human chorionic gonadotropin treated rats. After the same prolonged human chorionic gonadotropin treatment the concentration of pregnenolone was increased in mitochondrial fractions of whole testis tissue, interstitial tissue and seminiferous tubules, and the amount of protein isolated in the mitochondrial fraction of interstitial tissue increased by 40%. Steroid production (estimated as pregnenolone) from endogenous precusors by mitochondrial fractions of whole testis tissue and interstitial tissue were increased after human chorionic gonadotropin treatment, for whole testis from 580 pmol/mg mitochondrial protein per h to 1420 pmol/mg per h; and for interstitial tissue from 2665 pmol/mg per h to 7050 pmol/mg per h. The production of pregnenolone in mitochondrial fractions obtaine from isolated seminiferous tubules was very low and contributed hardly at all to the total pregnenolone production in mitochondrial fractions of whole testis tissue from normal rats as well as from human chorionic gonadotropin-treated rats.     

Immunolocalization of ecto-5'-nucleotidase in the kidney by a monoclonal antibody

A monoclonal antibody IgG, has been raised against ecto-5'-nucleotidase purified from rat kidney homogenate. The specificity of the antibody was verified by immunoprecipitation. The distribution of the corresponding antigen in the rat kidney was studied by immunocytochemistry (FITC and PAP technique) in 1 micron thick cryostat sections. The antibody reacted with the brush border of proximal tubules, the apical cell membrane and the apical cytoplasm of intercalated cells in connecting tubules and collecting ducts and with interstitial cells of the cortex. Among the interstitial cells exclusively stellate shaped fibroblasts were reactive whereas rounded interstitial cells (type II interstitial cells) as well as pericytes and endothelial cells of peritubular capillaries were unreactive. Compared to the staining intensity of the fibroblasts in the cortical labyrinth the reactivity of the fibroblasts in the medullary rays of the cortex was weak or absent. Interstitial cells of the entire medulla were unreactive. Concerning the fibroblasts in the periarterial connective tissue, those surrounding the larger arteries (arcuate arteries, cortical radial arteries) were negative, those alongside afferent and efferent arterioles were positive. Endothelia of lymphatic capillaries travelling within the periarterial connective tissue were also positive. All components of the juxtaglomerular apparatus were negative. The findings are consistent with an interstitial production of adenosine, available extracellularly and thus being able to reach the major target sites of adenosine, the smooth muscles of glomerular arterioles, including the granular cells at the glomerular vascular pole.     

Regulation of human monocyte matrix metalloproteinases by SPARC

SPARC (secreted protein, acidic and rich in cysteine), also called osteonectin or BM-40, is a collagen-binding glycoprotein secreted by a variety of cells and is associated with functional responses involving tissue remodeling, cell movement and proliferation. Because SPARC and monocytes/macrophages are prevalent at sites of inflammation and remodeling in which there is connective tissue turnover, we examined the effect of SPARC on monocyte matrix metalloproteinase (MMP) production. Treatment of human peripheral blood monocytes with SPARC stimulated the production of gelatinase B (MMP-9) and interstitial collagenase (MMP-1). Experiments with synthetic peptides indicated that peptide 3.2, belonging to the alpha helical domain III of SPARC, is the major peptide mediating the MMP production by monocytes. SPARC and peptide 3.2 were also shown to induce prostaglandin synthase (PGHS)-2 as determined by Western and Northern blot analyses. The increase in PGHS-2 stimulated by SPARC or peptide 3.2 correlated with substantially elevated levels of prostaglandin E₂ (PGE₂) and other arachidonic acid metabolites as measured by radioimmunoassay and high performance liquid chromatography (HPLC), respectively. Moreover, the synthesis of MMP was dependent on the generation of PGE₂ by PGHS-2, since indomethacin inhibited the production of these enzymes and their synthesis was restored by addition of exogenous PGE₂ or dibutyryl cAMP (Bt₂cAMP). These results demonstrate that SPARC might play a significant role in the modulation of connective tissue turnover due to its stimulation of PGHS-2 and the subsequent release of PGE₂, a pathway that leads to the production of MMP by monocytes. J. Cell. Physiol. 173:327–334, 1997. Published 1997 Wiley-Liss, Inc.

1 This article was prepared by a group of United States government employees and non-United States government employees, and as such is subject to 17 U.S.C. Sec. 105.

     

Interleukin-6 induces the synthesis of tissue inhibitor of metalloproteinases-1/erythroid potentiating activity (TIMP-1/EPA).

This study examines the role of interleukin-6 (IL-6) in connective tissue metabolism. Effects of different preparations of IL-6 on production of collagenase and tissue inhibitor of metalloproteinases-1/erythroid potentiating activity production are studied in human fibroblasts, synoviocytes, and articular chondrocytes. In contrast to interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha), IL-6 does not stimulate the production of collagenase, nor does it modulate the stimulatory effects of IL-1 beta and TNF alpha on the production of this proteinase. Furthermore, IL-6 has no detectable effect on prostaglandin E2 production, an additional proinflammatory response induced by IL-1 beta and TNF alpha. IL-6, however, is identified as a potent inducer of de novo synthesis of tissue inhibitor of metalloproteinases-1/erythroid potentiating activity in all types of connective tissue cells examined. These results define new biological activities of IL-6 and provide further insight into the regulation of connective tissues by cytokines.     

Synaptic activity and connective tissue remodeling in denervated frog muscle

Denervation of skeletal muscle results in dramatic remodeling of the cellular and molecular composition of the muscle connective tissue. This remodeling is concentrated in muscle near neuromuscular junctions and involves the accumulation of interstitial cells and several extracellular matrix molecules. Given the role of extracellular matrix in neurite outgrowth and synaptogenesis, we predict that this remodeling of the junctional connective tissue directly influences the regeneration of the neuromuscular junction. As one step toward understanding the role of this denervation-induced remodeling in synapse formation, we have begun to look for the signals that are involved in initiating the junctional accumulations of interstitial cells and matrix molecules. Here, the role of muscle inactivity as a signal was examined. The distributions of interstitial cells, fibronectin, and tenascin were determined in muscles inactivated by presynaptic blockade of muscle activity with tetrodotoxin. We found that blockade of muscle activity for up to 4 wk produced neither the junctional accumulation of interstitial cells nor the junctional concentrations of tenascin and fibronectin normally present in denervated frog muscle. In contrast, the muscle inactivity induced the extrajunctional appearance of two synapse-specific molecules, the acetylcholine receptor and a muscle fiber antigen, mAb 3B6. These results demonstrate that the remodeling of the junctional connective tissue in response to nerve injury is a unique response of muscle to denervation in that it is initiated by a mechanism that is independent of muscle activity. Thus connective tissue remodeling in denervated skeletal muscle may be induced by signals released from or associated with the nerve other than the evoked release of neurotransmitter.     

The frequency of chromosome aberrations in rat bone marrow cells in the late period after a single uptake of tritium

A prolonged self-maintenance of haemopoietic tissue cells with stable chromosome rearrangements following a single intake of tritium oxide in the amount of 24 MBq/g of body weight (absorbed dose of 11 Gy) is shown. Mutant cells revealed long after the radionuclide exposure are descendants of stem-cell precursors, bearing stable chromosome aberrations during the period of formation of radiation injury after the radionuclide administration.     

Is the meniscus of the knee joint a fibrocartilage?

A histological analysis of the structure of intact knee joint menisci was carried out in adult dogs. By means of specific histochemical methods for the connective tissue and cartilage, it was found that the meniscus as a whole does not have a unique structure. The anterior and posterior horns are populated by round chondroid cells encircled by abundant interstitial substance and branched wavy connective fibers; blood vessels are present. The outer third of the meniscus is constituted of cross bundles of connective fibers, fibrocytes and spindle-like areas of loose connective tissue with blood vessels. The inner avascular two thirds of the meniscus are filled with parallel circumferentially oriented fascicles of connective fibers, ovally elongated chondroid cells, and a small quantity of chondroid interstitial substance. In some menisci, in the inner two thirds of the body, there are isles of typical cartilage, which show metachromasia of the beta type and rarely of the gamma type. The occurrence and way of the manifestation of cartilage are of an individual character. The structural duality of the knee meniscus is accounted for by its functional duality manifested in offering resistance to the forces of traction and pressure, the latter ones favoring the process of evolution of tissue from connective, through chondroid, to cartilaginous.     

Demonstration of prostaglandin synthesis in collecting duct cells and other cell types of the rabbit renal medulla.

The renal medulla has a high capacity for prostaglandin production and the interstitial cells, which contain abundant lipid inclusions have been suggested to be the site of synthesis. However, histochemical studies have indicated that the collecting ducts are the main site of production. The object of the present study was to study the distribution of prostaglandin synthetase in the rabbit renal medulla by direct, quantitative determination of the enzyme activity in different cellular fractions. Slices were cut from rabbit renal papilla and immersed in a hypertonic saline solution. 92% of the collecting duct cells were then removed from the slices by suction through a micropipette. The remaining dissected slices thus contained mainly three cell types, cells of Henle's loop, endothelial cells, and interstitial cells. The isolated collecting duct fraction, the corresponding dissected slices, from which the colelcting duct cells were removed, as well as intact slices were assayed for prostaglandin synthetase activity using a quantitative assay with [14C] arachidonate as substrate. Of the prostaglandin in synthetase activity 39% was found in the collecting ducts, 53% in the dissected slices, and 7% in the dissection medium. It is thus concluded that significant prostaglandin synthetase activity is present in collecting duct cells as well as in at least one other cell type of the medulla.     

Effects of isolation and culture on prostaglandin synthesis by porcine aortic endothelial and smooth muscle cells

Freshly isolated neonatal porcine aortic tissue (smooth muscle with or without endothelium present) produced approximately 30 ng/mg wet tissue of 6-oxo-prostaglandin F1 alpha (the stable hydrolysis product from prostacyclin) and approximately 15 ng/mg of prostaglandin E2, as measured by radioimmunoassay after 24 h incubation in culture medium. Primary cultures of porcine endothelial and smooth muscle cells (isolated by enzymic digestion of aortic tissue) exhibited the same pattern of prostaglandin production, but absolute values were greater than for fresh tissue, particularly in the case of endothelium. Subcultures of endothelium produced smaller amounts of prostaglandins, although the pattern remained similar. In contrast, subcultures of smooth muscle cells produced a greater total amount of prostaglandins than did primary cultures, and the main product was prostaglandin E2. Experiments with [14C] prostaglandin H2 or [14C]arachidonic acid confirmed that aortic tissue, cultured endothelium, and primary cultures or aortic smooth muscle cells synthesized prostacyclin, and demonstrated that subcultured smooth muscle cells enzymically isomerised prostaglandin H2 to prostaglandin E2. Kinetic studies showed that prostaglandin production by cultured vascular cells was transiently increased by subculture or changing the growth medium, and that production per cell declined with increasing cell density. The change in pattern of prostaglandin production during culture was shown to be due to a rapid decline in the rate of prostacyclin production (which apparently began immediately after tissue isolation), together with a more gradual rise in prostaglandin E2 production. These results indicate that the amounts and ratios of prostaglandins produced by vascular endothelial and smooth muscle cells are greatly affected by the conditions used to isolate and culture the cells; vascular cells in vivo may similarly alter their pattern of prostaglandin production in response to local changes in their environment.     

Selective destruction and regeneration of rat Leydig cells in vivo

Summary The effect of a single i.p. administration of ethane dimethanesulphonate (EDS) upon rat testicular histology was studied by light microscopy and morphometry up to 4 weeks after treatment. One day after injection the interstitial tissue exhibited degenerating Leydig cells, abundant pyknotic interstitial cells, deposition of cellular debris and extensive networks of fibrillar material. Macrophages contained greatly increased numbers of cytoplasmic inclusion bodies. From 3 to 7 days morphometric analysis showed that Leydig cells and cellular debris had disappeared from the interstitial tissue, leaving only macrophages, fibroblasts and lymphatic endothelial tissue. A very small number of new Leydig cells were seen on day 14, often located in peritubular or perivascular positions. Regeneration of foetal-like Leydig cells occurred by 4 weeks, their cytoplasm containing large lipid inclusions and, numerous Leydig cells were often observed closely applied to the walls of the seminiferous tubules. The observations suggest that, after experimental destruction and depletion of Leydig cells, an interstitial precursor cell, as yet unidentified, gives rise to a new Leydig cell population. EDS thus offers a valuable opportunity to study further the interactions between the seminiferous tubules and the interstitial tissue following the destruction and subsequent regeneration of the Leydig cells.     

Demonstration of prostaglandin synthesis in collecting duct cells and other cell types of the rabbit renal medulla

The renal medulla has a high capacity for prostaglandin production and the interstitial cells, which contain abundant lipid inclusions, have been suggested to be the site of synthesis. However, histochemical studies have indicated that the collecting ducts are the main site of production. The object of the present study was to study the distribution of prostaglandin synthetase in the rabbit renal medulla by direct, quantitative determination of the enzyme activity in different cellular fractions.Slices were cut from rabbit renal papilla and immersed in a hypertonic saline solution. 92% of the collecting duct cells were then removed from the slices by suction through a micropipette. The remaining dissected slices thus contained mainly three cell types, cells of Henle's loop, endothelial cells, and interstitial cells. The isolated collecting duct fraction, the corresponding dissected slices, from whcih the collecting duct cells were removed, as well as intact slices were assayed for prostaglandin synthetase activity using a quantitative assay with [¹⁴C] arachidonate as substrate.Of the prostaglandin synthetase activity 39% was found in teh collecting ducts, 53% in the dissected slices, and 7% in the dissection medium. It is thus concluded that significant prostaglandin synthetase activity is present in collecting duct cells as well as in at least one other cell type of the medulla.