S100-like protein in the goldfish (Carassius auratus) kidney

The presence and distribution of S100-like protein in the goldfish (Carassius auratus L.) kidney has been studied by the use of immunohistochemical and histochemical methods. Simple immunohistochemistry (peroxidase anti-peroxidase method) was carried out with a polyclonal antibody against a mixture of both S100alpha and S100beta proteins. In order to confirm the cell-type containing S-100-like immunoreactivity, the colocalization of S-100-like protein immunoreactivity with periodic acid-Schiff (PAS) reaction was investigated by using double staining with indirect immunofluorescence and PAS histochemistry. S100-like immunoreactivity was detected only in juxtaglomerular cells located in the renal arterial branch and never on afferent arterioles. No immunoreactivity was observed in other tracts of the nephron or in the interstitial cells. Double staining confirmed that S-100-like immunoreactivity and PAS reactivity were colocalized in juxtaglomerular cells. These findings are the first regarding the presence and distribution of S100-like protein in the teleost kidney; they add a new member to the list of extra-neural S100-like-containing cell types and confirm that the antigen cannot be regarded as nervous-system-specific. In addition, a concentration of S100-like immunoreactivity in juxtaglomerular cells suggests the presence of S100-like calcium-binding protein-mediated activities in these cell types.     

Studies on the juxtaglomerular apparatus

The juxtaglomerular apparatus of the rat was studied after freeze-fracturing with special respect to intercellular junctions. It was found that juxtaglomerular granulated cells of the vas afferens are interconnected by gap junctions to adjacent cells (granulated cells, possibly also smooth muscle cells). Gap junctions have also been found on the surface of lacis cells and mesangial cells. It is therefore concluded that these cells of the juxtaglomerular apparatus and the glomerulus--granulated cells (possibly also smooth muscle cells) of the vas afferens, lacis cells and mesangium cells--form a functional system reacting in a coordinated manner to physiological stimuli.     

Granulation rescue and developmental marking of juxtaglomerular cells using "piggy-BAC" recombination of the mouse ren locus

Mice lacking a functional Ren-1(d) gene exhibit a complete lack of renal juxtaglomerular cell granulation and atypical macula densa morphology. Transgenic mice carrying a 145-kilobase BAC clone encompassing the Ren-1(d) and Ren-2 loci were generated, characterized, and backcrossed with Ren-1(d-/-) mice. Homozygous Ren-1(d)-null mice expressing the BAC clone exhibited complete restoration of normal renal structure. Homologous recombination in Escherichia coli was used to generate a modified version of the BAC clone, in which an IRESbeta-geo cassette was inserted specifically into the Ren-1(d) gene. When introduced into the germline, the modified clone provided a marker for juxtaglomerular cell differentiation and beta-geo was expressed appropriately in juxtaglomerular cells throughout development. Parallel backcross experiments onto the Ren-1(d)-null background demonstrated that the juxtaglomerular cells expressed the modified Ren-1(d) locus in the absence of regranulation. These data demonstrate that the nongranulated cells constitute bona fide juxtaglomerular cells despite their altered morphology, that overexpression of renin-2 cannot compensate for the loss of renin-1(d), and that primary structural differences between the two isoforms are responsible for the differences in granulation. The use of BAC modification as part of functional complementation studies illustrates the potential for in vivo molecular dissection of key physiological mechanisms.     

Ultrastructure of the juxtaglomerular apparatus of the kidney and of the peripolar cells in the sand lizard (Lacerta agilis)

In 9 sand lizards ultrastructure of the juxtaglomerular complex of the kidney has been studied. It is presented as juxtaglomerular cells, situating in the middle tunic of the afferent glomerular arteriole near the vascular pole of the renal corpuscle. Cytoplasm of these cells contains secretory granules at various stages of development: young, maturing and mature, as well as solid corpuscles and myofilaments. In some nephrons primitive forms of the macula densa and the juxtaglomerular island occur. Their presence demonstrates phylogenetically new structural organization of the juxtaglomerular complex in lizards. For the first time in reptilia peripolar cells are found, they are situated on the basal membrane of the external part of the glomerular capsule near the vascular pole of the renal corpuscle. A suggestion is made on their functional interconnection with the juxtaglomerular complex.     

Changes in expression of inhibin subunits in the cyclic golden hamster (Mesocricetus auratus) and the regulation of inhibin alpha subunit production by luteinizing hormone

In the present study, changes in localization of each inhibin subunit in the ovary were investigated during the estrous cycle of the golden hamster. The effect of LH surge on changes in localization in inhibin alpha subunit in the ovary was also investigated. Inhibin alpha subunit was localized in granulosa cells of various stages of follicles throughout the estrous cycle. Inhibin alpha subunit was also present in numerous interstitial cells on days 1 and 2 (day 1 = day of ovulation), but the number of positive interstitial cells was fewer on days 3 and almost disappeared on day 4 of the estrous cycle. Newly formed luteal cells were also positive for inhibin alpha subunit on days 1 and 2. On the other hand, positive reactions for inhibin beta A and beta B subunits were only present in the granulosa cells of healthy antral follicles. However, a positive reaction for inhibin beta B subunit in peripheral mural granulosa cells disappeared on days 3 and 4 of the estrous cycle. Treatment with LHRH-AS at 1100 h on day 4 completely blocked the luteinizing hormone (LH) surge and ovulation, although relatively high concentrations of plasma follicle-stimulating hormone (FSH) were maintained throughout the experiment. There were few positive reactions for inhibin alpha subunit in theca and interstitial cells 24 hr after LHRH-AS injection. The effect of LHRH-AS treatment was blocked by a single injection of 10 IU human chorionic gonadotropin. These results suggest that the major source of dimeric inhibin in the cyclic hamster was granulosa cells of healthy antral follicles. Different distribution pattern of inhibin beta A from beta B subunits in large antral follicles on days 3 and 4 of the estrous cycle suggests different secretion patterns of inhibin A from B on these days. Furthermore, the LH surge may be an important factor to induce production of inhibin alpha subunit in interstitial cells of the cyclic hamster.     

Single alpha-domain constructs of the Na+/Ca2+ exchanger, NCLX, oligomerize to form a functional exchanger

Spliced isoforms of the Na+/Ca2+ exchanger, NCLX, truncated at the alpha-repeat region have been identified. The activity and functional organization of such proteins are, however, poorly understood. In the present work, we have studied Na+/Ca2+ exchange mediated by single alpha-repeat constructs (alpha1 and alpha2) of NCLX. Sodium-dependent calcium transport was fluorescently detected in both the reversal and forward modes; calcium-dependent outward currents were also recorded using a whole cell patch configuration in HEK293 cells heterologously expressing either the alpha1 or alpha2 single-domain proteins. In contrast, calcium transport and reversal currents were not detected when cells were transfected with a vector or with an alpha2 mutant (alpha2-S273T). Thus, our data indicate that the single alpha-domain constructs mediate electrogenic Na+/Ca2+ exchange. The alpha1 domain, but not the alpha2, exhibited partial sensitivity to the NCX inhibitor, KB-R7943, while Li+-dependent Ca2+ efflux was detected in cells expressing either the alpha1 or alpha2 construct. The functional organization of the single alpha-domain constructs was assessed using a dominant-negative approach. Coexpression of the alpha1 or alpha2 constructs with the nonfunctional alpha2-S273T mutant had a synergistic inhibitory effect on Na+/Ca2+ transport. Dose-dependence analysis of the inhibition of alpha2 construct activity by the alpha2-S273T mutant indicated that the functional unit is either a dimer or a trimer. Immunoprecipitation analysis indicated that the alpha2 construct indeed interacts with the alpha2-S273T mutant. Taken together, our data indicate that although single alpha1 or alpha2 domain constructs are independently capable of Na+/Ca2+ exchange, oligomerization is required for their activity. Such organization may give rise to transport activity with distinct kinetic parameters and physiological roles.     

Ultrastructural studies on the secretory process in the epithelioid cells of the juxtaglomerular apparatus

The epithelioid cells of the juxtaglomerular apparatus have been studied with respect to the release mechanism of the secretory granules. Invaginations of the plasma membrane into the interior of the epithelioid cells are interpreted as stages before or after an exocytotic process. Granules are sometimes observed in close contact with the plasma membrane, and material with electron density similar to that of the granules can also be observed in the invaginations. These morphological features suggest that the granular material of the epithelioid cells is extruded into the texture of the basal lamina. Furthermore, a dense network of microtubules and microfilaments is described and the functional role of this system in exocytosis is discussed.     

Characteristics of interstitial cells of the adrenal medulla after stimulation of prostaglandin production by lasix

The paper presents structural characteristics of interstitial cells after a single or multiple administration of lasix. The single dose stimulated the process of prostaglandins synthesis in the interstitial cells of Kidney papillae. The prolonged administration of lasix led to relative decrease in prostaglandin production if compared with that of a single dose. Besides, there was noted formation of these cells second population, which elaborated the connective tissue proteins and glycosaminoglycans. It was suggested that the prolonged administration of lasix effected the interstitial cells causing inanition of their prostaglandin synthesizing ability and stimulating processes of production of the connective tissue intercellular substances by them.     

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.     

S-100 alpha-like immunoreactivity in tubules of rat kidney. A clue to the function of a "brain-specific" protein

The localization of the alpha and beta subunits of S-100 protein was studied in normal tissue where the identification of three subclasses of S-100 containing cells was derived: i) cells that contain both alpha and beta subunits; ii) cells that contain only the alpha subunit; and iii) cells that contain only the beta subunit. In this study monospecific antibodies against the S-100 alpha and beta subunits were used to characterize the S-100-like immunoreactivity in the rat kidney: Certain cells in the distal nephron, i.e., the connecting piece, collecting ducts, and the thin limb of Henle's loop, contained S-100 alpha immunoreactivity. Proximal tubules, the thick ascending limb of Henle's loop, the distal tubules, and the juxtaglomerular apparatus were negative. No S-100 beta immunoreactivity was found in kidney tubules. However, Schwann cells of renal pelvic nerves contained S-100 beta immunoreactivity. The presence of S-100 alpha antigen in certain cells of the kidney gives further support to the assumption that the alpha subunit of S-100a is related to cells that are highly involved in pH, electrolyte, and water regulation.     

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.     

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

Summary 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 m 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.     

Immunocytochemical and biochemical studies on the localization and changes of 17 alpha-hydroxylase/C17-C20 lyase activity in immature rat ovary treated with PMSG and hCG

The localization of cytochrome P-450 of 17 alpha-hydroxylase/C17-C20 lyase (P-450(17 alpha, lyase] and the changes of the enzyme activity were studied immunocytochemically and biochemically in the ovaries of immature rats treated with PMSG (pregnant mare serum gonadotropin) and hCG (human chorionic gonadotropin). Immunocytochemically, P-450(17 alpha, lyase) was localized in both the theca interna cells and interstitial gland cells of the ovaries of immature rats treated with PMSG for 48 h. After hCG administration, the immunoreactive cells rapidly decreased in number in the PMSG-pretreated rat ovary. Namely, 6 h after the hCG injection, positive staining for P-450(17 alpha, lyase) was recognized only in a few theca interna cells, while 12 h after the injection to immunostained cells were detected in the ovary. Forty-eight hours after the hGC treatment (96 h after the PMSG injection), most of the theca interna cells and the interstitial gland cells became immunopositive for P-450(17 alpha, lyase) again. The 17 alpha-hydroxylating activity of P-450(17 alpha, lyase) was 0.5, 0.22 and 0.03 nmol/min/mg protein in the ovarian microsomes of PMSG-treated, PMSG + hCG(3 h)-treated and PMSG + hCG(6 h)-treated rats, respectively. Changes of the hydroxylase activities in all the experimental groups are almost parallel to those of P-450 contents in the microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the juxtaglomerular apparatus

The vascular pole of the juxtaglomerular apparatus in Tupaia belangeri was studied with special reference to the intercellular contacts of the periendothelial cells and the endothelium of the vas afferens. The periendothelial cells of the vascular pole of the glomerulum are connected by numerous gap junctions; and the granulated epithelial cells are suggested to form a functional unit. Probably there is a continuity of this system throughout the entire vascular pole including (1) all granulated cells, (2) all lacis cells, (3) the mesangium cells and (4) the adjacent smooth muscle cells of the vas afferens and vas efferens. Analysis of the endothelial junctions shows a zonular arrangement of tight junctions indicating a rather tight blood-tissue barrier next to the glomerular vascular pole; The ultrastructure of the different cell types of the vas afferens is also described, emphasizing the granulated epithelial cells and their innervation.     

Subset of cells immunopositive for neurokinin-1 receptor identified as arterial interstitial cells of Cajal in human large arteries

In the adventitia of large arteries, dendritic cells are located between nerve fibers, some of which contain substance P. The aim of the present study was to examine whether neurokinin 1 receptor (NK-1R) was expressed by dendritic cells in the arterial wall. Parallel sections of aortic and carotid artery segments were immunostained with anti-NK-1R and cell-type-specific antibodies. Dendritic cells in the arterial wall expressed NK-1R, albeit at a low level. Other cells, which intensely expressed NK-1R, were located along the border between the media and adventitia. They did not co-express any dendritic cell markers, including fascin, CD1a, S100, or Lag-antigen, and were negative for CD68, CD3, and mast cell tryptase. These NK-1R⁺ cells were laser-capture microdissected and studied by means of electron-microscopic analysis. The microdissected cells were in direct contact with nerve endings, and their ultrastructure was typical of the interstitial cells of Cajal present in the gastrointestinal tract. Further systematic electron-microscopic analysis revealed that the cells displaying the features typical of interstitial cells of Cajal were a basic element of the human arterial wall architectonics. Arterial interstitial cells of Cajal were negative for c-kit but they expressed vasoactive intestinal peptide receptor 1 (VIPR1). Destructive alterations of contacts between arterial interstitial cells of Cajal and nerve endings were observed in arterial segments with atherosclerotic lesions. The functional significance of the arterial interstitial cells of Cajal and their possible involvement in atherosclerosis and other vascular diseases need clarification.This work was supported by the St Vincents Clinic Foundation, Sydney, Australia.     

Prostaglandin inhibition of testosterone production induced by luteinizing hormone, dibutyryl cyclic AMP or 3-isobutyl-1-methyl xanthine in dispersed rat testicular interstitial cells.

Testicular interstitial cells were utilized to study the effects of prostaglandins (PG) on in vitro testosterone production and to examine the role of cyclic adenosine-3',5'-monophosphate (cAMP) in this process. Testosterone production was assessed after 3 hour incubations while cAMP accumulation was examined after a 0.5 hour incubation period. Testosterone and cAMP were measured by radioimmunoassay. None of the PGs tested (PGA, PGA2, PGB1, PGE1, PGE2, PGF1alpha PGF2alpha) altered basal testosterone production when present in incubates at concentrations of 1.3 X 10(-8) M to 1.3 X 10(-4). However, at concentrations of 1.3 X 10(-4) M all of these PGs were capable of decreasing Luteinizing Hormone (LH; 100ng)-induced testosterone production. The inhibition of LH-induced testosterone production by the B, E and F series PGs was less pronounced than that for the A series. PGA1 and PGA2 exhibited 80% and 95% inhibition, respectively, at 1.3 X 10(4) M. The inhibitory action of 4 X 10(5) M PGA1 or PGA2 was evident within 30 minutes. Preincubation of interstitial cells with indomethacin (10(-5) or 10(-6) M) for 30 minutes did not alter subsequent basal or LH (100ng)-induced testosterone production. Accumulation of cAMP was stimulated by LH (10 microgram) or by PGs (1.3 X 10(-4) M PGA1, PGA2, PGB1, PGE1 or PGF2alpha). The PG-induced cAMP accumulation thus occurred at concentrations where LH-stimulated testosterone production was inhibited. Furthermore, PGA1 and PGA2 (1.3 X 10(-4) M) inhibited testosterone production induced by either 3-isobutyl-1-methyl xanthine (MIX; 10(-4) M or 10(-3) M) or dibutyryl cAMP (dbcAMP; 10(-4) M or 10(-3) M). These results indicate that PGs can block testosterone production by a direct effect on testicular interstitial cells and suggest that PGs exert their inhibitory action distal to stimulation of cAMP formation. PGs do not appear to play a role in the mechanism of LH action.     

Juxtaglomerular apparatus and interstitial cells of the kidney medulla after the administration of certain drugs and use of hyperbaric oxygenation

Electron microscopy was used to examine the status of the juxtaglomerular apparatus (JGA) and interstitial cells (IC) 3 and 24 hours after administration of furosemide (10 mg/kg), indomethacin (10 mg/kg), venoruton (500 mg/kg) and trental (100 mg/kg), and after 1,6 an 12 sessions of hyperbaric oxygenation. To evaluate objectively the results of examining the JGA, use was made of a method devised by the authors of a mathematical appraisal of granulation from the density of the epithelioid cells. Granulation of 50 IC from each animal was calculated on semi-thin araldite sections stained methylene blue-azur II-fuchsin. The results indicate that all the types of exposure including hyperbaric oxygenation produced JGA activation whose degree varied depending on the time elapsed after exposure. An apparently great increase in the JGA activity was detected after injection of furosemide and indomethacin. All the drugs with the exception of furosemide entailed granule accumulation after 3 hours, followed by the recovery of their amount after 24 hours. Furosemide injection produced a reverse effect.     

Modulation of intracellular Ca2+ via L-type calcium channels in heart cells by the autoantibody directed against the second extracellular loop of the alpha1-adrenoceptors

The effects of methoxamine, a selective alpha1-adrenergic receptor agonist, and the autoantibody directed against the second extracellular loop of alpha1-adrenoceptors were studied on intracellular free Ca2+ levels using confocal microscopy and ionic currents using the whole-cell patch clamp technique in single cells of 10-day-old embryonic chick and 20-week-old fetal human hearts. We observed that like methoxamine, the autoantibody directed against the second extracellular loop of alpha1-adrenoreceptors significantly increased the L-type calcium current (I(Ca(L))) but had no effect on the T-type calcium current (I(Ca(T))), the delayed outward potassium current, or the fast sodium current. This effect of the autoantibody was prevented by a prestimulation of the receptors with methoxamine and vice versa. Moreover, treating the cells with prazosin, a selective alpha1-adrenergic receptor antagonist blocked the methoxamine and the autoantibody-induced increase in I(Ca(L)), respectively. In absence of prazosin, both methoxamine and the autoantibody showed a substantial enhancement in the frequency of cell contraction and that of the concomitant cytosolic and nuclear free Ca2+ variations. The subsequent addition of nifedipine, a specific L-type Ca2+ channel blocker, reversed not only the methoxamine or the autoantibody-induced effect but also completely abolished cell contraction. These results demonstrated that functional alpha1-adrenoceptors exist in both 10-day-old embryonic chick and 20-week-old human fetal hearts and that the autoantibody directed against the second extracellular loop of this type of receptors plays an important role in stimulating their activity via activation of L-type calcium channels. This loop seems to have a functional significance by being the target of alpha1-receptor agonists like methoxamine.