Intracellular Renin Research Articles

Mechanisms of Disease: local renin–angiotensin–aldosterone systems and the pathogenesis and treatment of cardiovascular disease

Accumulating evidence has made it clear that not only does the renin-angiotensin-aldosterone system (RAAS) exist in the circulation where it is driven by renal renin, but it is also active in many tissues-and likely within cells as well. These systems might not be completely independent of each other, but rather interact. These local RAASs affect tissue and cellular angiotensin II concentrations and appear to be associated with clinically relevant physiologic and pathophysiologic actions in the cardiovascular system and elsewhere. Evidence in support of this possibility is reviewed here. In addition, direct (pro)renin action after binding to its specific receptor, the existence of renin transcripts, which apparently encode an intracellular renin, the discovery of an angiotensin-converting-enzyme homologue (ACE2), which leads to enhanced generation of angiotensin-(1-7) and the newly appreciated role of angiotensin-receptor dimerization in the regulation of angiotensin activity, all point to the conclusion that the RAASs are complexly regulated, multifunctional systems with important roles both within and outside the cardiovascular system.

High glucose concentration stimulates intracellular renin activity and angiotensin II generation in rat mesangial cells.

Increased intrarenal renin-angiotensin system activity contributes to diabetic nephropathy. ANG II generation in mesangial cells (MC) is increased by high-glucose (HG) exposure. This study assessed the mechanisms involved in the glucose-induced ANG II generation in rat MC. Under basal conditions, MC mainly secreted prorenin. HG decreased prorenin secretion and induced a striking 30-fold increase in intracellular renin activity. After 72 h of HG exposure, only the mRNA levels for angiotensinogen and angiotensin-converting enzyme (ACE) were significantly elevated. However, after shorter periods of 24 h of HG stimulation the mRNA levels of the enzymes prorenin and cathepsin B, besides that for ACE, were significantly increased. The results suggest that the HG-induced increase in ANG II generation in MC results from an increase in intracellular renin activity mediated by at least three factors: a time-dependent stimulation of (pro)renin gene transcription, a reduction in prorenin enzyme secretion, and an increased rate of conversion of prorenin to active renin, probably mediated by cathepsin B. The increase in angiotensinogen mRNA in parallel to increased renin activity indicates that HG also increased the availability of the renin substrate. The consistent upregulation of ACE mRNA suggests that, besides renin, ACE is directly involved in the increased mesangial ANG II generation induced by HG.

Intracellular renin and the nature of intracrine enzymes.

Recently, the binding of renin and prorenin to cellular receptors with the subsequent generation of second messengers and the production of physiological effects has been demonstrated. In addition, the internalization of prorenin by target cells has been associated with increased cellular synthesis of angiotensin and cardiac pathology. Also, a renin transcript lacking the sequences encoding a secretory signal has been reported, and this transcript appears to produce a renin that acts in the cell that synthesized it. Some years ago, we coined the term intracrine for a peptide hormone or factor that acts in the intracellular space either after internalization or retention in its cell of synthesis. Thus defined, a wide variety of peptides display intracrine functionality, including hormones, growth factors, transcription factors, and enzymes. For example, considerable evidence indicates that angiotensin II is an intracrine. Also, general principles of intracrine functionality have been developed. Thus, recent evidence demonstrates that the prorenin/renin molecule is an intracrine enzyme. Here, the actions of intracrine enzymes (angiogenin, phosphoglucose isomerase, phospholipase A2, granzyme A and B, thioredoxin, platelet-derived endothelial growth factor, and serine protease inhibitors) are reviewed. The relation of prorenin/renin to other intracrine enzymes, and to intracrines in general, is discussed.

Regulation of renin: new evidence from cultured cells and genetically modified mice.

Renin, as the rate-limiting enzyme in the synthesis of the potent vasoactive peptide angiotensin II, has been studied for more than 100 years. Transgenic and knockout mice for renin and other proteins involved in renin regulation and function have recently revealed new evidence that can improve our understanding of its biological relevance. Furthermore, transgenic mice have been the source of the novel cell line As4.1. This cell line has been effective in the analysis of renin secretion and regulation because of its similarity with renin-producing juxtaglomerular (JG) cells. Renin secretion and synthesis by the JG cells of the kidney is upregulated by cAMP and downregulated by intracellular calcium. The effect of cGMP, once elevated by nitric oxide, depends on the present level of cAMP in the cells, which can be stimulatory in the presence of and inhibitory in the absence of the other cyclic nucleotides. All known effectors of renin regulation affect one of these molecules. Adenosine and ATP, released by macula densa cells in response to high salt load in the distal tubule and stretch of the JG cell by renal perfusion pressure, increase calcium. Furthermore, noradrenaline, derived from sympathetic nerve endings, and prostaglandins, generated by macula densa cells under low-salt conditions, increase cAMP. In addition to its stimulatory effect on secretion, cAMP also effectively augments renin mRNA levels by acting at the transcriptional and posttranscriptional levels. Several DNA elements in the distal and proximal promoter regions as well as in intron I have been implicated in cAMP regulation and in tissue specificity of renin gene expression. A second intracellular renin isoform, coded by the same gene but applying a different promoter located in intron I, has recently been detected. Transgenic technology will help to clarify the function of this isoform as well as some of the other unresolved aspects of renin regulation and function and may become the motor of the second century in renin research.

Novel Perspectives on Pituitary and Brain Angiotensinogen

All the angiotensin peptides originate from angiotensinogen, a glycoprotein synthesized by several tissues, including the brain and the anterior pituitary. In the rat, immunohistochemistry has been used to localize angiotensinogen in gonadotropes and in uncharacterized cells surrounding sinusoids. Both cell types are capable of secreting angiotensingen in cell culture; only the gonadotropes contain angiotensin II (AngII) and are capable of secreting it in culture. It has been asserted that the perisinusoidal cells are the only source of angiotensinogen for the generation of AngII by gonadotropes. Our current data favor the existence of a complete intracellular renin–angiotensin system (RAS) in gonadotropes and a separate extracellular system which utilizes the high concentration of angiotensinogen from perisinusoidal cells. Furthermore, we postulate that gonadotrope AngII serves mainly reproductive functions, while the proximity of angiotensinogen-secreting cells to folliculostellate cells, and their access to the intercellular sinusoidal and follicular spaces, places the extracellular RAS in a strategic position to affect pituitary growth and the mediation of acute-phase immune responses. In the rat brain, angiotensinogen is expressed by the 16–18th day of fetal life and by areas generally concerned with vasopressor, electrolyte, and fluid homeostasis. Antisense deoxyoligonucleotides to angiotensinogen mRNA lower blood pressure in hypertensive rats and inhibitin vitrogrowth of neuroblastoma cells, indicating a significant role for angiotensinogen in mitogenic and homeostatic functions. It is commonly agreed that astrocytes express angiotensinogen. Neuronal angiotensinogen has also been demonstrated by immunohistochemistry, as a secretion from neuronal cell cultures, and by reverse-transcriptase polymerase chain reaction. The fate of secreted astrocytic and neuronal angiotensinogen remains obscure. Angiotensinogen is regulated in a tissue-specific manner with smaller or absent responses observed for brain tissue. By using astrocyte and neuronal cultures the actions on angiotensinogen production of growth hormone, IGF-1, inflammatory lipopolysaccharide, and phorbol ester have been examined. Recent observations show that angiotensinogen is regulated positively or negatively by glucocorticoids and that a positive synergism between cAMP and glucocorticoids exists. On the basis of analogous systems for other proteins, a scheme involving glucocorticoid receptors, CREB, and AP-1 transcription factors is formulated to explain glucocorticoid–cAMP interactions. These transcriptional interactions may form a significant functional link between the RAS and adrenergic mechanisms.

Biosynthesis of renin in mouse kidney tumor As4.1 cells.

As4.1, a renin-expressing cell line isolated from a mouse renal tumor, was characterized for synthesis, processing, storage and secretion of renin polypeptides. Metabolic labeling, immunoprecipitation and SDS/PAGE analysis revealed that renin was secreted into the culture supernatant predominantly in the form of prorenin which migrated as products of 42-47 kDa. The predominant intracellular renin was processed into two chains, of 33-34 and 5 kDa. N-glycanase treatment removed N-linked oligosaccharides and yielded products of 41 kDa for prorenin and 31-32 kDa for the heavier chain of two-chain renin. The N-terminus of the constitutively secreted prorenin was determined by automated Edman degradation to be Leu22 while the N-terminus of the heavy chain was Ser72. Renin polypeptides constituted 3.1 +/- 1.4% (mean percentage of total precipitable radioactivity +/- SD) of de-novo-synthesized protein secreted into the medium and 0.2 +/- 0.17% retained intracellularly. Extrapolation of renin activity assays suggest that a single cell stores approximately 680 fg of active renin. A slow incremental release into the medium of processed renin heavy chain was detected by immunoprecipitation and SDS/PAGE. Renin activity assays confirmed the release of approximately 4 fg prorenin and 0.32 fg active renin cell(-1) h(-1). Indirect immunofluorescence demonstrated intracellular renin to be distributed in a punctate pattern. Renin was found to be colocalized with the lysosomal marker, beta-glucuronidase, by double-fluorescent labeling. These cells have enabled characterization of glycosylated mouse renin-1 and may prove a valuable tool for studying intracellular trafficing of renin and associated processing enzymes.

Renin Regulation in Cultured Proximal Tubular Cells

Recent studies have documented the presence of a complete renin-angiotensin system in the proximal tubule of the kidney: however, little is known about the regulation of renin in this proximal tubular system. Therefore, we performed the present studies to learn whether the behavior of the renin system in cultured proximal tubule is similar to that of the juxtaglomerular renin system. Basal renin secretion from rabbit proximal tubular cells in primary culture was low and not affected by isoproterenol (10(-5) mol/L), diltiazem (10(-5) mol/L), or a zero-calcium bath (O nmol/L). Only the calcium ionophore A23187 (10(-4) mol/L) significantly reduced renin secretion in these cells (from 2.44 +/- 0.37 to 1.14 +/- O.08 ng angiotensin I/mg protein per hour, P<.05). When the proximal tubular cells were lysed so the effects of the test agents on intracellular renin content could be assessed, isoproterenol caused a significant twofold (107 percent) increase (from 2.02 +/- 0.56 to 4.18 +/- 0.81 ng angiotensin I/mg protein per hour, P<.05), whereas diltiazem, A23187, and zero- and high-calcium baths did not produce a significant change. The effects of these agents on renin mRNA were examined in rabbit and rat proximal tubular cells in primary culture with the use of an S1 nuclease protection assay. Densitometry analysis of renin mRNA and either GAPDH mRNA (rat) or alpha-actin (rabbit) showed no significant alterations in renin mRNA abundance. In summary, these results confirm the presence of renin mRNA in cultured proximal tubular cells and suggest that a low-level, constitutive secretion of renin occurs in this system that is decreased by A23187. Moreover, the results also suggest that proximal tubular renin is regulated, albeit differently from the juxtaglomerular renin system. Finally, short-term increments in proximal tubular renin occur without a change in renin mRNA.

NEWBORN MESENTERIC ARTERY CAN PRODUCE ANGIOTENSIN-II (A-II) FROM RENIN SUBSTRATE (RS) OR ANGIOTENSIN-I • 1387

Initially, the renin-angiotensin system (RAS) was believed to be systemic. RS production and conversion to A-I occurred in the kidney; thereafter, conversion of A-I to A-II occurred in the lungs, followed by systemic distribution and action of the active octapeptide A-II. It has recently been reported that a complete RAS also exists within some endothelial cells (EC), ie., EC are capable of de novo RS production followed by local conversion to A-I and A-II by intracellular (ie., EC) renin and extracellular angiotensin converting enzyme (ACE ectoenzyme). To determine if the newborn gut is imbued with an EC-RAS, rings of mesenteric artery from 3-d old swine were mounted for isometric tension measurement in myographs filled with warm(38°C) oxygenated Krebs buffer and were administered RS, A-I or A-II (all 10-8M), +/- captopril (CAP, an ACE inhibitor, 10-5M) or losartan(LOS, an A-II AT1 receptor blocker, 10-7M). Rings were intact(EC+) or had the EC mechanically removed (EC-). Each ring was administered only one agonist or agonist+blocking agent. Contractile responses were expressed as a% of the maximal tension developed after KCI depolarization. A 3-way ANOVA was conducted on data from each agonist (EC+ vs EC-;% contraction vs 0%; agonist vs agonist+blocker), with post-hoc. Data (M±SEM, n=5 subjects):Table Elimination of the contractile response to RS or A-I following ACE-inhibition or A-II AT1 receptor blockade indicates that these agonist exerted their effects by local conversion to A-II, ie., that renin and ACE were present in the rings. Lack of response to RS or A-I in EC- indicates that the enzymes are located on the EC, whereas thesite of A-II effect is smooth muscle. Conclusion: A local RAS is present in the EC of mesenteric artery from 3-d old swine.Speculation: Locally-produced A-II may contribute tointrinsic vasoregulation of the newborn gut, ie., intrinsic constriction in response to systemic circulatory perturbations.

Influence of Intracellular Renin on Heart Cell Communication

The influence of intracellular renin and angiotensinogen on the control of cell-to-cell communication in heart muscle was investigated in cell pairs isolated from adult rat ventricle. Junctional conductance was measured with two separated voltage-clamp circuits. Intracellular dialysis of renin (0.2 pmol/L) caused a decrease in junctional conductance of 29 +/- 3.8% (+/- SEM, P < .05) in 7 minutes. The effect of renin on junctional conductance seems to be mainly due to the synthesis of Ang II because enalaprilat (10(-9) mol/L) dialyzed into the cell caused an appreciable reduction in the effect of renin. The intracellular administration of renin (0.2 pmol/L) plus angiotensinogen (0.4 pmol/L) produced a faster and stronger fall in junctional conductance (84.3 +/- 1.35%, P < .05), and the effect was greatly reduced by enalaprilat. The effects of both renin and angiotensinogen on junctional conductance were not related to a fall in surface cell membrane resistance or a change in series resistance. The effect of renin on junctional conductance was blocked by intracellular administration of a renin inhibitor (S 2864). Moreover, renin dialyzed into just one cell of the pair induced rectification of the junctional membrane, which was prevented by enalaprilat. The results support the view that an intracrine renin-angiotensin system in the heart regulates intercellular communication.

Is an intracellular renin-angiotensin system involved in control of cell communication in heart?

The possible influence of an intracellular renin-angiotensin system (RAS) on control of cell communication in heart muscle was investigated in cell pairs isolated from adult rats. Junctional conductance (gj) was measured with two separated voltage-clamp circuits. Intracellular dialysis of angiotensin I (AI 10(-8) M) caused a decrease in gj of 76% (SE +/- 3.4) (p < 0.05) in 7 min. The effect of AI appears to be due mainly to its conversion to AII because enalaprilat (10(-9) M) dialysed into the cell caused an appreciable reduction in the effect of AI. AII (10(-8) M) alone caused a decrease in gj of 60% (SE +/- 3.8) (p < 0.05) in 45 s. The effect of AII on gj was suppressed by previous inhibition of protein kinase C (PKC), but enalaprilat could not alter the effect of the peptide. The results indicate that synthesis of AII inside cardiac myocytes plays an important role in modulation of gj and consequently on propagation of the electrical impulse in heart. The effect of AII on gj was blocked by DuP-753 (10(-9) M) administered intracellularly, whereas (Sar1Val5AlA8) AII also caused a slight decrease (1.97 +/- 0.07%) in gj. These findings indicate that an intracellular receptor is involved in the effect of the peptide on gj.

Studies on the renin-angiotensin system in primary monolayer cell cultures of the rat epididymis.

Monolayer cell cultures formed from the rat cauda epididymidis exhibited renin-like and angiotensin-converting enzyme (ACE) activities and contained immunoreactive angiotensin I (AI) and angiotensin II (AII). Renin-like activity, determined indirectly by radioimmunoassay of generated AI at a near-neutral pH of 6.0, was demonstrated in the cell lysate but was almost undetectable in the serum-free cell culture medium, suggesting that renin expression in epididymal cells is an intracellular phenomenon. In contrast, both AI and AII were detected in the cell lysate and cell culture medium. The level of AI was enhanced by pretreating the cells with the ACE inhibitor captopril (100 nmol/l). Incubating the cell monolayers with thoroughly washed sperm cells obtained from the intact cauda epididymides of rats increase (P less than 0.01) the AII content of the cell culture medium, with a parallel decline (P less than 0.01) in the AI concentration. However, adrenaline (0.23 mumol/l), which was found to stimulate electrogenic anion secretion by cell monolayers grown on previous supports, was without effect on the renin-like activity or concentration of angiotensins. The ACE activity in cells was confirmed by its strong dependence on chloride ion and its susceptibility to inhibition by captopril (100 nmol/l). Enzyme activity was significantly (P less than 0.005) higher in the culture medium than in the cell lysate and cell membrane fragments. Angiotensinogen, which is obligatory for an intrinsic renin-angiotensin system, is present in epididymal cells. Presumably, it is synthesized and processed in the cell cytosol by intracellular renin.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation by hCG of ovarian inactive renin synthesis in rabbit preovulatory theca cells

The origin of ovarian renin and its regulation by hCG were investigated in rabbit periovulatory follicles and cultured preovulatory follicular cells. Intracellular content of renin in thecal cells was 8-fold greater than of granulosa cells. In vivo, administration of hCG increased intracellular content of renin in thecal but not granulosa cells. Similar results were obtained for cultured follicular cells, from which renin was partly released into the medium. In vitro, hCG increased intracellular renin content of thecal but not granulosa cells, without obvious effect on release. Approximately 95% of ovarian renin was inactive, but could be activated by trypsin. Thecal renin was antagonized in vitro by renin antiserum, indicating a specific renin activity. Our study establishes in the rabbit the thecal cell origin of ovarian inactive renin and demonstrates hCG regulation of its synthesis.

Modulation of renin synthesis by lipoxygenase products in cultured human mesangial cells

Modulation of renin synthesis by lipoxygenase products has been studied in cultured human mesangial cells under basal conditions and in the presence of prostaglandin (PG) E 2. Total renin and cyclic AMP productions were stimulated in a dose-dependent manner (0.1–10 μM) by PGE 2. The stimulatory effect of PGE 2 on renin production was inhibited by 12-hydroxyeicosatetraenoic acid (12-HETE) between 0.1 and 100 nM. Extracellular and intracellular renin were affected similarly. Neither basal and PGE 2-dependent cyclic AMP nor basal cyclic GMP productions were modified. 15-Hydroxyeicosatetraenoic acid (15-HETE), 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and 15-hydroperoxyeicosatetraenoic acid (15-HPETE) had the same effects as 12-HETE. Intracellular calcium concentration was not modified in the presence of 12-HETE. Since oleyl-2-acetylglycerol (OAG), an analog of diacylglycerol, also inhibited PGE2-stimulated renin production, it is hypothesized that the effect of the lipoxygenase products is mediated via protein kinase C stimulation.

Cyclosporine A enhances renin secretion and production in isolated juxtaglomerular cells

Stimulation of the renin-angiotensin system is a major side effect of the fungoid immunosuppressant cyclosporine A (CyA). The aim of this study was to find out whether or not this effect of CyA results from a direct interaction with renal juxtaglomerular (JG) cells, which are the site of renal renin synthesis and release. Using primary cell cultures from rat renal cortex containing more than 80% JG cells, we found that CyA (0.01 to 10 micrograms/ml) stimulated renin secretion threefold. This stimulation was paralleled by a dose-dependent twofold increase of inactive renin within the cells, while the active intracellular renin remained the same. In order to identify a possible second messenger which could mediate the effects of CyA on JG cells, we examined the simultaneous effects of a single concentration of CyA (1 microgram/ml) on renin secretion, prostaglandin formation and intracellular cAMP concentration. However, prostaglandin formation and cAMP were not detectably altered by CyA in experiments where renin secretion was significantly enhanced. Our results indicate that cyclosporine A stimulates renin secretion and renin synthesis by a direct effect on renal juxtaglomerular cells. This action of CyA is not mediated by changes in cellular prostaglandin or intracellular cAMP.

New possibilities for intracellular renin and inactive renin now that the structure of the human renin gene has been elucidated.

Other| October 01 1986 New possibilities for intracellular renin and inactive renin now that the structure of the human renin gene has been elucidated Brian J. Morris Brian J. Morris 1Molecular Biology and Hypertension Laboratory, Department of Physiology, The University of Sydney, New South Wales, Australia Search for other works by this author on: This Site PubMed Google Scholar Clin Sci (Lond) (1986) 71 (4): 345–355. https://doi.org/10.1042/cs0710345 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation Brian J. Morris; New possibilities for intracellular renin and inactive renin now that the structure of the human renin gene has been elucidated. Clin Sci (Lond) 1 October 1986; 71 (4): 345–355. doi: https://doi.org/10.1042/cs0710345 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsClinical Science Search Advanced Search This content is only available as a PDF. © 1986 The Biochemical Society and the Medical Research Society1986 Article PDF first page preview Close Modal You do not currently have access to this content.

Cultured Glomerular Mesangial Cells Contain Renin

Glomerular mesangial cells are contractile cells responsive to a variety of vasoactive substances. In addition, they are capable of synthesizing prostaglandins and renin-like enzyme(s) (RLE). We examined the identity of the RLE in cultured rat mesangial cells utilizing specific antibody raised to pure renal renin. Unlike cathepsin D, RLE is active at pH 7.4. One million mesangial cells contain 174 +/- 53 pg ANG I/h RLE intracellularly (ANG I, angiotensin I; n = 26). As evidenced by inhibition by renin-specific antibody, 52 +/- 3% RLE is due to immunoreactive renin. Mesangial immunoreactive renin activity is influenced by beta-adrenergic stimulation and increased extracellular calcium. Exposure to 1 microM isoproterenol at 37 degrees C for 1 h resulted in 103 +/- 53% increase in intracellular activity, i.e., 56 +/- 8 to 102 +/- 15 pg ANG I/h/10(6) cells (p less than 0.05, n = 7). Addition of calcium to culture media for 1 h resulted in an increase in intracellular renin activity. Addition of 1 mM (final concentration) calcium resulted in a ninefold increase in mesangial renin activity from 21 +/- 8 to 185 +/- 10 pg ANG I/h/10(5) cells (n = 4, p less than 0.001). Similarly, 4 mM calcium resulted in a sevenfold increase (n = 4, p less than 0.001). Thus, mesangial cells synthesize renin, which can be regulated by beta-adrenergic receptors and extracellular calcium. This intracellular renin may play an important role in the local regulation of contractile response and glomerular dynamics.

Endogenous renin inhibitor in neuroblastoma cells

Cloned neuroblastoma cells (Neuro-2a) in culture were found to contain a renin inhibitory substance. The inhibitor in the extract of cloned neuroblastoma cells was separated from renin activity by anti-renin IgG-Sepharose and selectively concentrated by adsorption to renin-agarose gel. The present study demonstrated the coexistence of renin and its inhibitor in the same cell and suggested a possible regulatory mechanism of intracellular renin activity by an endogenous renin inhibitor in neuronal cells.

Immunohistochemical evidence that angiotensins I and II are formed by intracellular mechanism in juxtaglomerular cells.

The existence of angiotensin II (AII) immunoreactivity in juxtaglomerular (JG) cells of rat kidney, which has been demonstrated previously by immunohistochemical studies, can be explained either as the product of intracellular synthesis or by the internalization of receptor-bound AII originating in plasma. To resolve these two alternative mechanisms, attempts were made to identify AI in JG cells of rat kidney by immunohistochemical staining using specific antibodies to AI. Although AI-like immunoreactivity was not detected in normal rat kidney, rats treated with the angiotensin-converting enzyme inhibitors, MK-421 or captopril, showed AI-like immunoreactivity in JG cells. The presence of renin and AII-like immunoreactivity was demonstrated in the same cells by specific antibodies to respective antigens used on adjacent serial sections. These findings support an intracellular mechanism of the formation of AII and suggest an intracellular renin angiotensin system, presumably separate from the extracellular system.

Renin and angiotensin: the complete system within the neuroblastoma x glioma cell.

Cells of the homogeneous hybrid line neuroblastoma x glioma (NG108-15) have many neuronal properties. Immunocytochemical tests show that they contain both immunoreactive renin and angiotensin; direct radioimmunoassays show that they are positive for renin, angiotensin I, and angiotensin II; enzymatic assays show that they contain angiotensinogen and converting enzyme as well. The renin appears to be present in an enzymatically inactive form that can be activated by trypsin and then blocked by antiserum to purified mouse submaxillary renin. Renin concentration and activity are increased by enhancing cellular differentiation with dibutyryl cyclic adenosine monophosphate or by serum withdrawal. These findings demonstrate a complete renin-angiotensin system within these neuron-like cells, and suggest that activation of intracellular renin could generate angiotensin II.

Effects of angiotensin II and angiotensin II antagonist saralasin on cell growth and renin in 3T3 and SV3T3 cells

Components of the renin-angiotensin system were studied in established cell culture lines of 3T3 and SV3T3 mouse fibroblasts. The renin content in 3T3 cells was significantly higher than in virus-transformed SV3T3 cells. With time after infection, renin decreased in Simian virus 40 transformed cells, while it increased steadily in mock-infected 3T3 cells. In contrast to renin, angiotensinase activity was higher in SV3T3 cells. Angiotensin II stimulated cell proliferation in 3T3 mouse fibroblasts and decreased their renin content in a dose-related manner. In contrast, saralasin, an angiotensin receptor antagonist, inhibited cell growth in 3T3 and SV3T3 cells and caused an increase of cellular renin concentration. The angiotensin fragments angiotensin (2-8) heptapeptide and angiotensin (4-8) pentapeptide had no effect on cell growth. A significant negative correlation was found between cell proliferation and renin levels in 3T3 and SV3T3 cells irrespective of the treatment. Our results indicate (1) that angiotensin II may be involved in cell growth regulation, (2) that a negative feedback exist between angiotensin II added and intracellular renin content, and (3) that virus infection causes a decrease in intracellular renin synthesis, while non-specific angiotensinase activity is increased under this condition.