Macroautophagic phenomena in renin granules

Summary The secretory granules of marine epithelioid cells take up and probably degrade mitochondria; they thus appear to have macroautophagic properties. As renin granules also have other properties uncommon for secretory granules, they are suggested to fulfill functions in these cells otherwise reserved for lysosomes.These studies were supported by the German Research Foundation within the Forschergruppe Niere/Heidelberg     

Are the renin-containing granules of juxtaglomerular epithelioid cells modified lysosomes?

Summary Mature secretory granules of epithelioid cells — the so-called renin granules — exhibit certain properties, which in this particular combination are expressed only by lysosomes: Renin granules have autophagic capabilities; they react to the application of lipidosis-inducing, lysosomotropic substances by the gradual accumulation of polar lipids; all secretory granules of epithelioid cells contain acid phosphatase until maturity; and exogenous tracers reach renin granules without labeling the Golgi complex. Several functional implications can therefore be considered. Hydrolytic enzymes, constitutive elements of the granule matrix, might either cleave inactive prorenin to yield active renin within the granules or, by unspecific hydrolysis of renin, participate in the regulation of the overall quantity of secretory product. Autophagic phenomena, the involvement of renin granules in the traffic of exogenous tracers, and the build-up of polar lipids following experimental interference with lipid catabolism indicate a large turnover of membrane material in renin granules. They also suggest that cytoplasmic and extracellular fluid gains access to the granule content and may thus be involved there in the regulation of biochemical reactions by changing the intragranular milieu or via signal molecules.In addition to the lysosome-like properties of epithelioid cell secretory granules, the secretory product, renin, as a carboxyl protease, is structurally related to other acidic proteases. In the case of cathepsin D, even functional similarities exist.These studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System.     

The fate of prorenin during granulopoiesis in epithelioid cells

Summary Comparative immunocytochemical experiments with antisera directed against renin and three synthetical peptides (Pro 1, Pro 2 A and Pro 3) covering almost the entire span of human renin prosegment were performed on human kidney tissue. With anti-Pro 1, i.e. the antiserum which recognizes the NH₂ terminus of human prorenin, no clear immunolabeling of juxtaglomerular epithelioid cell secretory granules could be obtained. It is therefore concluded that the corresponding portion of human prorenin may be cleaved off in the Golgi complex.After application of anti-Pro 3, the antiserum which recognizes the COOH terminus of the prosegment, only the juvenile secretory granules of epithelioid cells were consistently labeled, whereas, in contrast, some of the intermediate and most of the mature secretory granules were anti-Pro 3-negative. As the immunoreactivity of mature renin increased remarkably from protogranules to mature secretory granules, it is suggested that the cleavage of the COOH terminus of the prosegment, i.e. the activation of renin, takes place in juvenile and intermediate granules during condensation of the enzyme.The immunoreactivity of Pro 2A, corresponding to the middle portion of the prosegment, disappeared in a some-what earlier stage of granulopoiesis than that of Pro 3. It is therefore concluded that the corresponding segmental cleavage, the result of which is a truncated version of intact prorenin, occurs in the protogranules of epithelioid cells.The data presented are consistent with the assumption that the secretion of active renin takes place by the exocytosis of mature secretory granules, while the secretion of inactive renin, which is a truncated version of intact prorenin, is mediated by the exocytosis of juvenile and intermediate granules.These studies were supported by the German Research Foundation within the Forschergruppe Niere/Heidelberg     

The fate of prorenin during granulopoiesis in epithelioid cells. Immunocytochemical experiments with antisera against renin and different portions of the renin prosegment

Comparative immunocytochemical experiments with antisera directed against renin and three synthetical peptides (Pro 1, Pro 2A and Pro 3) covering almost the entire span of human renin prosegment were performed on human kidney tissue. With anti-Pro 1, i.e. the antiserum which recognizes the NH2 terminus of human prorenin, no clear immunolabeling of juxtaglomerular epithelioid cell secretory granules could be obtained. It is therefore concluded that the corresponding portion of human prorenin may be cleaved off in the Golgi complex. After application of anti-Pro 3, the antiserum which recognizes the COOH terminus of the prosegment, only the juvenile secretory granules of epithelioid cells were consistently labeled, whereas, in contrast, some of the intermediate and most of the mature secretory granules were anti-Pro 3-negative. As the immunoreactivity of mature renin increased remarkably from protogranules to mature secretory granules, it is suggested that the cleavage of the COOH terminus of the prosegment, i.e. the activation of renin, takes place in juvenile and intermediate granules during condensation of the enzyme. The immunoreactivity of Pro 2A, corresponding to the middle portion of the prosegment, disappeared in a somewhat earlier stage of granulopoiesis than that of Pro 3. It is therefore concluded that the corresponding segmental cleavage, the result of which is a truncated version of intact prorenin, occurs in the protogranules of epithelioid cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coexistence of renin and cathepsin B in epithelioid cell secretory granules

Summary Mature juxtaglomerular epithelioid cell secretory granules of the rat exhibit both renin- and cathepsin B-like immunoreactivity. On the basis of the coexistence with renin at a pH which, according to previous experiments, is probably in the range of that in lysosomes, cathepsin B is suggested to be involved in the activation of renin prior to secretion.These studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System     

Cathepsin D coexists with renin in the secretory granules of juxtaglomerular epithelioid cells

Summary Mature juxtaglomerular epithelioid cell secretory granules of the rat exhibit both renin-and cathepsin D-like immunoreactivity. On the basis of the coexistence with renin at a pH which, according to previous experiments, is probably in the range of that in lysosomes, cathepsin D is suggested to be involved in the regulation of the granular renin stores available for secretion.These studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System     

Hypothetical interpretation of the calcium paradox in renin secretion

Summary Most renin-positive cells of the preglomerular arteriole are intermediate in morphological appearence between smooth muscle cells and epithelioid cells. Intermediate cells contain, in addition to secretory granules, contractile proteins arranged as a sublemmal network. The paradoxical (inhibitory) role of calcium in renin secretion is explained, on the basis of these findings, by an increased tone of the sublemmal network; this might impair the preexocytotic access of renin granules to the cell membrane.This study was supported by the Deutsche Forschungsgemeinschaft within the Forschergruppe Niere, Heidelberg     

Renin processing studied by immunogold localization of prorenin and renin in granular juxtaglomerular cells in mice treated with enalapril

Summary Immunogold techniques were used to investigate renin processing within granular juxtaglomerular cells following short-term (6 h and 1 day) and long-term (4 weeks) enalapril treatment in female BALB/c mice. In control animals, renin protein labelling was localized to all types of granules (proto-, polymorphous, intermediate and mature) and to transport vesicles, whilst prorenin labelling was found in all these sites except mature granules, confirming that active renin is localized to mature granules only. Following short-term enalapril treatment, the exocytosis of renin protein from mature granules was increased. Long-term enalapril treatment resulted in increased numbers of transport vesicles and all types of granules, consistent with increased synthesis and storage of renin. More large intermediate granules contained discrete regions labelled for prorenin. Renin protein was exocytosed from individual and multiple granules, whilst prorenin was exocytosed from protoand intermediate granules. It is concluded that under normal conditions prorenin is secreted constitutively by bulk flow from transport vesicles. On the other hand, active renin is secreted regulatively from mature granules. In conditions of intense stimulation (angiotensin-converting enzyme inhibition treatment), increased synthesis of prorenin leads to enhanced secretion of prorenin by both constitutive and regulative pathways. Under these conditions, the conversion of prorenin to active renin is increased, with increased secretion of active renin occurring in a regulative manner. Furthermore, the localization of prorenin to one discrete region of large intermediate granules leads us to conclude, that cleavage of the prosegment of renin occurs with the transition of intermediate to mature granules.     

Effects of extracellular osmolality on renin release and on the ultrastructure of the juxtaglomerular epithelioid cell granules

Summary Superfusion with hypoosmotic solutions stimulates renin release from rat epithelioid cells adherent to isolated glomeruli. This stimulatory effect may be related to the observed swelling of the secretory granules; the swelling may markedly increase the probability of pre-exocytotic fusions between the granule and cell membranes, and consequently increase the frequency of exocytotic events.These studies were supported by the Danish Medical Research Council King Christian X's Foundation and the German Research Foundation within the Forschergruppe Niere/Heidelberg     

Immunocytochemical localization of cathepsin B in rat anterior pituitary endocrine cells, with special reference to its co-localization with renin and prorenin in gonadotrophs

We examined by immunocytochemistry the localization of cathepsin B in endocrine cells of rat anterior pituitary lobe, using a monospecific antibody to cathepsin B. By light microscopy, granular immunodeposits for cathepsin B were detected in most endocrine cells of anterior pituitary lobe. Cells immunoreactive for luteinizing hormone (LH) were diffusely immunostained by anti-cathepsin B. By electron microscopy, immunogold particles for cathepsin B were localized in lysosomes of thyrotrophs, somatotrophs, and mammotrophs. In mammotrophs, immunogold particles for cathepsin B were also detected in crinophagic bodies. Double immunostaining co-localized immunogold particles for LH and cathepsin B in secretory granules of gonadotrophs. Immunocytochemistry was also applied to demonstrate localization of renin and prorenin in LH-producing gonadotrophs; immunogold particles for renin were co-localized with those for LH, cathepsin B, or prorenin in their secretory granules. Immunogold particles for prorenin were also co-localized with those for LH or cathepsin B in secretory granules, but prorenin-positive granules appeared less frequently than renin-positive granules. These results suggest that cathepsin B not only plays a role in the protein degradation in lysosomes of anterior pituitary endocrine cells but also participates in the activation of renin in gonadotrophs, as has been demonstrated in secretory granules of juxtaglomerular cells.     

Coexistence of renin and angiotensin II in epitheloid cell secretory granules of rat kidney

Summary The distribution of renin and angiotensin II (ANG II) in juxtaglomerular epitheloid cells of control and adrenalectomized rats was studied, using specific antisera and the protein A-gold technique in Lowicryl- and glycol methacrylate-embedded tissue.The matrix of virtually all mature secretory granules of epitheloid cells contains not only renin, but also ANG II. On adrenalectomy, the concentration of both renin and ANG II in the granule internum increases markedly, as indicated by the density of the immunolabel.Given the coexistence of renin and ANG II in the granule matrix, it is quite probable that, with each secretory event, a certain amount of ANG II is released together with renin. Further experiments will have to show if this amount of ANG II cosecreted with renin is sufficient to elicit immediate local intrarenal actions.ANG I, as well as angiotensinogen and converting enzyme, were not found in epitheloid cells. It is therefore inferred that ANG II is not generated intracellularly, but within the extracellular space and subsequently taken up by pinocytosis and incorporated into the secretory granules of epitheloid cells.These studies were supported by the Deutsche Forschungsgemeinschaft within the SFB 90 Cardiovasculäres System     

Myosin content and vasoconstrictive ability of the proximal and distal (renin-positive) segments of the preglomerular arteriole

Summary The PAP-technique and antibodies to myosin were used to demonstrate the prerequisites for vasoconstriction in the juxtaglomerular part of the preglomerular arteriole as compared with its proximal segment in rats and mice. In contrast with the myosin-positive/renin-negative proximal part of the afferent arteriole no myosin-like activity could be demonstrated in its distal, renin-positive part. In accordance, no thick myofilaments were found in fully differentiated juxtaglomerular epithelioid cells replete with mature secretory granules. Stimulation of the renin-angiotensin system was followed by an increase of the reninpositive/myosin-negative portions of the preglomerular arteriole. Marked interspecies and internephron variations in the length of this vessel segment under control and stimulated conditions were observed.The juxtaglomerular part of the preglomerular arteriole close to the macula densa seems therefore to have only limited capabilities for vasoconstriction. This finding may be of importance regarding the tubulo-glomerular feedback, a mechanism allegedly triggered by the so-called macula densa-signal. It is suggested that this non-contractile segment of the afferent arteriole may represent the renal vascular receptor responsible for the increase of renin secretion during pressure reduction.Unlike the afferent arterioles, most of the efferent arterioles showed the highest level of their weak but distinct myosin-like immunoreactivity in the juxtaglomerular region, indicating some efferent juxtaglomerular vasoconstrictive ability.These studies were supported by the German Research Foundation within the Forschergruppe Niere/Heidelberg     

Recombinant human prorenin from CHO cells: Expression and purification

The gene for human preprorenin was obtained from total RNA prepared from primary human chorion cells. An expression vector was constructed containing an SV40 early promoter, a human preprorenin cDNA, bovine growth hormone poly-A addition signal, and a dihydrofolate reductase (dhfr) expression cassette. This vector was inserted into the DXB-11 Chinese hamster ovary (CHO) cell line. The recombinant protein was exported by CHO cells into the tissue culture media. At harvest the prorenin levels ranged from 1–5 mg/L. For prorenin isolation the cell culture supernatants were processed by filtration, concentration, dialysis, and batch extraction. Preparative-scale isolation of prorenin was accomplished using blue-dye chromatography and size-exclusion chromatography. The isolated prorenin yielded a single SDS-gel band with Mr 40,000. The proprotein was characterized with respect to N-terminal sequence and N-linked sugar composition. Trypsin-activated renin prepared from the proprotein was characterized with respect to N-terminal sequence andpH-activity profile. Enzyme activity was measured with a newly developed fluorogenic peptide substrate containing the P₆-P₃ sequence of human angiotensinogen.     

Renin activation in juvenile secretory granules?

Summary In immunocytochemical experiments on human kidney tissue with an antiserum directed against the prosegment of renin, only juvenile granules were clearly labeled. As the concentration of renin increases from protogranules to more mature granules, while the concentration of its prosegment decreases to subthreshold levels, it is assumed that the cleavage of the prosegment, i.e. the activation of renin takes place in juvenile granules parallel to the condensation of the enzyme.These studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System     

Über Aktivitätsphasen im Epithel des Saccus vasculosus von Etmopterus spinax

Summary The coronet cells in the saccus vasculosus of Etmopterus spinax L. were investigated histologically and histochemically. Inclusions of granula in the coronet cells were observed in 8 specimens. The granules react positivly as lipid and protein containing substances. They are attached to the Einschlußkörper in the cell base and accumulate around the nuclei or in the inner fibre apparatus of the cell apex. The diameter of the granules reaches 2,5 in the cell base and diminishes to less than 1 in the apical structures. These granula are interpreted as a sign of secretion into the third ventricle.This secretory activity is high in gravid females (31,4–54,7 % of the cells) and low in juvenile females or specimens in which no ovulation could be stated (0–4,7 %). Gravid females show differences in the distribution pattern of the granula within the cells, thus indicating a phasic activity.The coronet cells in the saccus vasculosus of Etmopterus seem to be involved in the secretory phenomena related to gravidity.

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Die Untersuchungen wurden mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft durchgeführt.     

Multiple rough endoplasmic cisternae in “C” cells

Summary Multiple rough endoplasmic cisternae were found in C cells of the adult rat, at interphase. They are considered to be normal constituents of C cells. Their morphological relation to rough endoplasmic reticulum and their close proximity to mitochondria, Golgi dictyosomes and secretory granules suggest that they may have a role in the secretory activity of this endocrine cell.     

Immunocytochemical localization of prorenin, renin, and cathepsins B, H, and L in juxtaglomerular cells of rat kidney

To examine the correlation of localization of prorenin, renin, and cathepsins B, H, and L, immunocytochemistry was applied to rat renal tissue, using a sequence-specific anti-body (anti-prorenin) that recognizes the COOH terminus of the rat renin prosegment. In serial semi-thin sections, immunodeposits for prorenin, renin, and cathepsins B, H, and L were localized in the same juxtaglomerular (JG) cells. Immunodeposits for renin were detected throughout the cytoplasm of the cells, whereas those for prorenin were detected in the perinuclear region. Immunoreactivity for cathepsin B was stronger than that for cathepsins H and L. By electron microscopy, prorenin was localized in small (immature) granules but not in large mature granules, whereas renin was localized mainly in mature granules. In serial thin sections, prorenin, renin, and cathepsin B were colocalized in the same immature granules containing heterogeneously dense material (intermediate granules). By double immunostaining, co-localization of renin with cathepsins B, H, or L was demonstrated in mature granules. The results suggest the possibility that processing of prorenin to renin occurs in immature granules of rat JG cells, and cathepsin B detected in JG cells may be a major candidate for the maturation of renin.     

Stereological study of gonadotropes in the frog,Rana pipiens,after GnRH stimulation in vitro

Summary Previous physiological results have indicated the existence of two releasable pools of gonadotropins in amphibian pituitaries: an acute releasable pool that appears independent of protein synthesis, and a storage pool involved in chronic release that depends on protein synthesis. To elucidate the ultrastructural localization of these pools and the morphological changes induced in gonadotrope cells after treatment with gonadotropin-releasing hormone, we carried out a morphometric study of immuno-identified gonadotrope cells using an in vitro superfusion system. Treatment with gonadotropin-releasing hormone induced a degranulation of small (110–255 nm) and medium (236–360 nm) secretory granules as well as hypertrophy of the endoplasmic reticulum and Golgi complex. Simultaneous incubation with gonadotropin-releasing hormone and cycloheximide inhibited the release of secretory granules although the endoplasmic reticulum and Golgi complex were hypertrophied. These morphological results strongly suggest: (1) that gonadotropin-releasing hormone induces degranulation and hypertrophy of the biosynthetic machinery in gonadotrope cells; and (2) that the activation of the endoplasmic reticulum and Golgi complex by stimulation with gonadotropin-releasing hormone is independent of protein synthesis, while the release of secretory granules is protein synthesis-dependent. In addition, the second or storage pool of gonadotropin is associated mainly with the small and medium secretory granules.     

Characterization of recombinant human renin: Kinetics,pH-stability, and peptidomimetic inhibitor binding

The kinetic behavior andpH-stability of recombinant human renin was analyzed using a new fluorogenic substrate based on the normal P₆-P₃ renin cleavage sequence in human angiotensinogen. The design of this fluorogenic substrate makes possible, for the first time, direct monitoring of the kinetics of proteolytic conversion of prorenin to renin. ThepH-stability profile for renin, measured with the substrate at 25°C, indicated a broad plateau of stability betweenpH 6.0 and 10.0. Analysis of thepH-activity profile of renin for the substrate indicated a minimumK _m (1.8 µM) atpH 7.4 and a maximumV _m betweenpH 7.4 and 8.0. The thermodynamics of the binding of a novel, soluble, peptidomimetic inhibitor to renin indicated it is possible to retain the tight-binding characteristics and enthalpy contributions to binding of larger peptide-derived inhibitors, while reducing inhibitor size and entropic contributions to binding. A novel derivative of the fluorogenic substrate, containing a 3-methyl histidine substitution at the P₂ site, was used to test the recent hypothesis that renin functions by virtue of substrate-directed catalysis.     

A targeting sequence for dense secretory granules resides in the active renin protein moiety of human preprorenin

Human renin plays an important role in blood pressure homeostasis and is secreted in a regulated manner from the juxtaglomerular apparatus of the kidney in response to various physiological stimuli. Many aspects of the regulated release of renin (including accurate processing of prorenin to renin, subcellular targeting of renin to dense secretory granules, and regulated release of active renin) can be reproduced in mouse pituitary AtT-20 cells transfected with a human preprorenin expression vector. Using protein engineering, we have attempted to define the roles of various structures in prorenin that affect its production and trafficking to dense core secretory granules, resulting in its activation and regulated secretion. Replacement of the native signal peptide of human preprorenin with that of a constitutively secreted protein (immunoglobulin M) had no apparent effect on either the constitutive secretion of prorenin or the regulated secretion of active renin in transfected AtT-20 cells. Removal of the pro segment resulted in a marked reduction in total renin secretion, but did not prevent renin from entering the regulated secretory pathway. Single or combined mutations in the two glycosylation sites of human renin did not prevent its regulated secretion; however, the complete elimination of glycosylation resulted in a significant increase in the ratio of renin/prorenin secreted by the transfected cells. Thus, these results suggest that 1) at least one of the sequences that target human renin to dense secretory granules lies within the protein moiety of active renin; 2) the presence of the pro segment is important for efficient prorenin and renin production; and 3) glycosylation can quantitatively affect the proportion of active renin secreted.