Prorenin Synthesis Research Articles

Kidney Angiotensin in Cardiovascular Disease: Formation and Drug Targeting.

The concept of local formation of angiotensin II in the kidney has changed over the last 10–15 years. Local synthesis of angiotensinogen in the proximal tubule has been proposed, combined with prorenin synthesis in the collecting duct. Binding of prorenin via the so-called (pro)renin receptor has been introduced, as well as megalin-mediated uptake of filtered plasma-derived renin-angiotensin system (RAS) components. Moreover, angiotensin metabolites other than angiotensin II [notably angiotensin-(1-7)] exist, and angiotensins exert their effects via three different receptors, of which angiotensin II type 2 and Mas receptors are considered renoprotective, possibly in a sex-specific manner, whereas angiotensin II type 1 (AT1) receptors are believed to be deleterious. Additionally, internalized angiotensin II may stimulate intracellular receptors. Angiotensin-converting enzyme 2 (ACE2) not only generates angiotensin-(1-7) but also acts as coronavirus receptor. Multiple, if not all, cardiovascular diseases involve the kidney RAS, with renal AT1 receptors often being claimed to exert a crucial role. Urinary RAS component levels, depending on filtration, reabsorption, and local release, are believed to reflect renal RAS activity. Finally, both existing drugs (RAS inhibitors, cyclooxygenase inhibitors) and novel drugs (angiotensin receptor/neprilysin inhibitors, sodium-glucose cotransporter-2 inhibitors, soluble ACE2) affect renal angiotensin formation, thereby displaying cardiovascular efficacy. Particular in the case of the latter three, an important question is to what degree they induce renoprotection (e.g., in a renal RAS-dependent manner). This review provides a unifying view, explaining not only how kidney angiotensin formation occurs and how it is affected by drugs but also why drugs are renoprotective when altering the renal RAS.Significance StatementAngiotensin formation in the kidney is widely accepted but little understood, and multiple, often contrasting concepts have been put forward over the last two decades. This paper offers a unifying view, simultaneously explaining how existing and novel drugs exert renoprotection by interfering with kidney angiotensin formation.

The Prorenin Receptor

The discovery of the prorenin receptor, now nearly a decade ago, altered our view of the renin-angiotensin system (RAS).[1][1],[2][2] Binding of prorenin, the inactive precursor of renin, to the prorenin receptor results in full catalytic activity of prorenin through a nonproteolytic mechanism that

Cardiac Renin Levels Are Not Influenced by the Amount of Resident Mast Cells

To investigate whether mast cells release renin in the heart, we studied renin and prorenin synthesis by such cells, using the human mast cell lines human mastocytoma 1 and LAD2, as well as fresh mast cells from mastocytosis patients. We also quantified the contribution of mast cells to cardiac renin levels in control and infarcted rat hearts. Human mastocytoma 1 cells contained and released angiotensin I-generating activity, and the inhibition of this activity by the renin inhibitor aliskiren was comparable to that of recombinant human renin. Prorenin activation with trypsin increased angiotensin I-generating activity in the medium only, suggesting release but not storage of prorenin. The adenylyl cyclase activator forskolin, the cAMP analogue 8-db-cAMP, and the degranulator compound 48/80 increased renin release without affecting prorenin. Angiotensin II blocked the forskolin-induced renin release. Angiotensin I-generating activity was undetectable in LAD2 cells and fresh mast cells. Nonperfused rat hearts contained angiotensin I-generating activity, and aliskiren blocked approximately 70% of this activity. A 30-minute buffer perfusion washed away >70% of the aliskiren-inhibitable angiotensin I-generating activity. Prolonged buffer perfusion or compound 48/80 did not decrease cardiac angiotensin I-generating activity further or induce angiotensin I-generating activity release in the perfusion buffer. Results in infarcted hearts were identical, despite the increased mast cell number in such hearts. In conclusion, human mastocytoma 1 cells release renin and prorenin, and the regulation of this release resembles that of renal renin. However, this is not a uniform property of all mast cells. Mast cells appear an unlikely source of renin in the heart, both under normal and pathophysiological conditions.

Critical Review of Prorenin and (Pro)renin Receptor Research

The renin-angiotensin system (RAS) plays an important role in cardiovascular and renal physiology and disease, and the benefits of angiotensin-converting enzyme inhibitor, angiotensin type 1 receptor blocker, and renin inhibitor therapies are mediated in part by their modification of the levels and actions of angiotensin peptides. Despite its long history, the RAS remains an active area of research. Contrary to the classical view of the RAS as an endocrine system whereby kidney-secreted renin acts on circulating angiotensinogen to produce angiotensin peptides in the circulation, it is now recognized that tissues are the main sites of angiotensin peptide formation by the action of plasma- (kidney-) derived renin on plasma-derived and locally synthesized angiotensinogen.1–3 A receptor for prorenin and renin was recently identified4,5 that is hereby referred to as the (pro)renin receptor. Prorenin and (pro)renin receptor research offers the exciting possibility of a new paradigm for the RAS whereby renin and prorenin binding to the (pro)renin receptor not only target and facilitate angiotensin generation but also lead to activation of (pro)renin receptor signal transduction pathways distinct from angiotensin II receptor signals.6,7 By revealing novel potential mechanisms of disease pathogenesis, this new paradigm offers the possibility of new therapies that may be more effective than those currently available. It also suggests that stimulation of the (pro)renin receptor by the increased renin and prorenin levels that accompany angiotensin-converting enzyme inhibitor, angiotensin type 1 receptor blocker, and renin inhibitor therapies may attenuate the benefits these therapies.7,8 Recent reviews and editorials have discussed this research.6,9,10 The purpose of this brief review is to critically assess the evidence for this new paradigm from the perspective of disease pathogenesis and to advocate caution in its interpretation. The available evidence presents contradictions and difficulties of interpretation that preclude any conclusion about the reality of …

Prorenin

Prorenin, the precursor of renin, exists in circulating blood at concentrations that are ≈5 to 10× higher than those of renin. For many years, prorenin was considered to be an inactive form of renin with no physiological role. Then, in the mid-80s of the last century, Luetscher et al reported that the levels of circulating prorenin (but not renin) are increased in diabetic subjects.1 Subsequent studies revealed that these high levels correlated with the presence of microvascular complications, and it was proposed that prorenin might be used to predict the occurrence of microalbuminuria.2 Simultaneously, high prorenin levels were observed in pregnant women, and evidence was obtained for the local synthesis of prorenin at extrarenal tissue sites such as the ovary and eye. Until today, however, the exact function of prorenin remains unknown. An attractive concept is that in tissues not synthesizing renin locally, circulating prorenin, after its local conversion to renin, contributes to angiotensin (Ang) generation. This would not only provide a role for prorenin in vivo, but also explain why tissues, in contrast with plasma, contain predominantly renin.3 Studies in transgenic animals support this idea. Veniant et al4 described that transgenic rats expressing prorenin exclusively in the liver (resulting in a 400-fold rise in plasma prorenin) display cardiac hypertrophy and vascular damage in the absence of hypertension. Prescott et al5 detected increased cardiac Ang I levels in mice overexpressing human prorenin in the liver and human angiotensinogen in the heart. The question then arises how tissues sequester and activate circulating prorenin. Sequestration may involve diffusion into the interstitial fluid and/or binding to a receptor.3 …

Renin, Prorenin and the Putative (Pro)renin Receptor

Renin is an aspartic protease that consists of 2 homologous lobes. The cleft in between contains the active site with 2 catalytic aspartic residues.1 Unlike other aspartic proteases such as pepsin or cathepsin D, renin is monospecific and only cleaves angiotensinogen, to generate angiotensin (Ang) I. Ang I is the precursor of the active end-product of the renin-angiotensin system (RAS), Ang II. Renin has also been called active renin to underline that an enzymatically inactive form of renin exists. In 1971, Lumbers found that amniotic fluid, left at low pH in the cold, acquired renin activity.2 Later, Skinner described a similar phenomenon in plasma.3 Acidification was not strictly necessary for this increase in Ang I-generating activity, because incubation at low temperature also increased renin activity, albeit to only 15% of activity after acidification. Soon it was postulated that this inactive, but activatable “big” renin (its molecular weight was 5 kDa higher than that of renin) was the biosynthetic precursor of renin. Hence, it was named prorenin. Only with the cloning of the renin gene in 1984 was prorenin definitively proved to be the precursor of renin.4 For reasons that are unknown, prorenin circulates in human plasma in excess to renin, sometimes at concentrations that are 100-times higher.5 Prorenin has also been demonstrated in plasma of cat, dog, cattle, pig, horse, sheep, rabbit, rat, and mouse. A 43-amino acid N-terminal propeptide explains the absence of enzymatic activity of prorenin. This propeptide covers the enzymatic cleft and obstructs access of angiotensinogen to the active site of renin. (Pro)renin is synthesized as a preprohormone. It contains a signal peptide that directs the protein to the endoplasmic reticulum and ultimately to the exterior of the cell. Both renin and prorenin can be fractionated into multiple species by isoelectric focusing. …

Follicular fluid renin concentration in patients with polycystic ovaries treated with gonadotrophins in an in vitro fertilisation programme.

The initial hypothesis was that the follicular fluid prorenin concentration was higher in women with polycystic ovaries (PCO) compared to normally cycling women stimulated by gonadotrophins. In a controlled prospective study, 47 women undergoing in vitro fertilisation (IVF) were enrolled: 20 women with PCO represented the study group and 27 normally cycling women the control group. Plasma prorenin, active renin, serum oestradiol, and androstenedione concentrations were measured on the day of human chorionic gonadotrophin (HCG) administration and 36 hours after HCG administration. Follicular fluid prorenin, active renin, oestradiol, and androstenedione concentrations from the dominant follicle and pooled follicles were measured 36 hours after HCG administration. Plasma prorenin concentration 36 hours after HCG administration was significantly higher in the PCO group (1867 microU/l; range 1137-3162 microU/l) than in controls (860 microU/l; range 433-1763 microU/l) (p = 0.007). The follicular fluid prorenin concentration in the dominant follicle and in the pooled follicles was lower in the PCO group (20190 microU/l; range 11130-25955 microU/l) than in controls (46930 microU/l; range 20671-66171 microU/l) (p = 0.003). We conclude that plasma prorenin concentrations in gonadotrophin-stimulated PCO cycles are augmented due to numerous follicles. Lower follicular fluid prorenin concentrations in women with PCO show that the synthesis of prorenin in theca cells starts later after HCG administration than in normally cycling women.

Stimulation of nitric oxide-cyclic guanosine monophosphate pathway in bovine ovarian theca cells by tumor necrosis factor alpha (TNF alpha). Is this pathway implicated in the TNF alpha-induced inhibition of luteinizing hormone-stimulated prorenin production?

Gonadal function is known to be controlled by many factors, including locally acting cytokines like tumor necrosis factor alpha (TNF alpha). One of the ways this cytokine acts is via the nitric oxide (NO)-cGMP pathway. Since we have shown that in the ovary theca cells are a target of TNF alpha's action, it was of interest to determine whether TNF alpha stimulates the NO-cGMP pathway in these cells and whether such a mechanism can be implicated in the observed TNF alpha-mediated inhibition of LH-stimulated prorenin synthesis and secretion. Treatment of isolated theca cells with TNF alpha resulted in a dose- and time-dependent increase in cGMP production. This increase was not detectable until 6 h after the addition of TNF alpha and was totally abolished by the protein synthesis inhibitor cycloheximide. Addition of either L-N6-nitroarginine methyl ester (L-NAME), an inhibitor of all three NO synthase (NOS) isoforms or 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), a specific inhibitor of the inducible isoform of the enzyme, likewise reversed the action of TNF alpha on cGMP formation. Finally, addition of 1H-[1,2,4]oxadiazolo [4,3-a] quinoxalin 1-one (ODQ), an inhibitor of NO-sensitive soluble guanylate cyclase, resulted in a concentration-dependent reduction of TNF alpha-stimulated cGMP formation. In contrast, the TNF alpha-mediated inhibition of LH-stimulated prorenin secretion was not affected by either L-NAME, AMT, or ODQ. Also the addition of stimulators of soluble guanylate cyclase, sodium nitroprusside, and S-nitroso-N-acetylpenicillamine, or 8 bromo-cGMP had no effect on the action of LH on theca cells. We conclude that although TNF alpha is able to stimulate cGMP formation in theca cells by inducing the expression of inducible NOS, the mechanism underlying the TNF alpha-mediated inhibition of LH-stimulated prorenin production is independent of its ability to induce cGMP formation.

β-Adrenoceptor Activation-Induced Placental Prorenin Secretion Is Mediated by Increased Renin Messenger RNA and Protein Synthesis

Activation of beta-adrenoceptors has been shown to promote renin secretion in both human kidney and placenta. In kidney, the enhanced secretion is immediately observed, and mobilization of renin in the storage granules accounts for such a rapid response. In contrast, the enhanced secretion in placenta is delayed for 6-12 hr after receptor activation and consists almost entirely of the renin precursor prorenin. It is hypothesized that newly synthesized rather than stored enzyme is responsible for the enhanced secretion in human placenta. To test this hypothesis, placental explants were cultured in the presence or absence of the protein synthesis inhibitor cycloheximide, and prorenin concentrations in the tissue and medium were measured. Dobutamine and terbutaline, beta1- and beta2-adrenoceptor agonists, evoked 17- and 5-fold increases in secretion, respectively. Tissue content of prorenin in response to the treatment was increased by a similar magnitude, yet values were consistently <10% of medium concentrations. The increases in prorenin concentrations in both medium and tissue, however, were markedly attenuated by cycloheximide, suggesting that prorenin synthesis in response to beta-adrenoceptor activation is required. Reverse transcription coupled with polymerase chain reaction revealed that renin mRNA levels were increased by 3-8-fold and occurred before increases in tissue and medium prorenin, indicating that increased renin mRNA levels are responsible for the increased synthesis of prorenin. Explants cultured in the presence of actinomycin D, an inhibitor of transcription, did not show the agonist-induced prorenin mRNA levels or enhancement of its secretion. The peak levels of renin mRNA were reached after 6 hr of incubation, were sustained at similar levels after 24 hr, and were not affected by cycloheximide. These findings are consistent with the notion that enhancement of renin mRNA and de novo protein synthesis are required for prorenin secretion induced by activation of placental beta-adrenoceptors.

Stimulation of placental prorenin secretion by selective inhibition of cyclic nucleotide phosphodiesterases

Prorenin secretion by human villous placenta is known to be stimulated by activation of adenylate cyclase and enhanced cyclic AMP (cAMP) generation. Placental tissue contains predominantly type III (cGMP-inhibited) and type IV (cAMP-specific) phosphodiesterases (PDEs), which inactivate cAMP. To evaluate the role of PDE subtypes in the regulation of prorenin secretion by human placenta, explants were cultured in the presence of isobutylmethylxanthine (IBMX), a non-selective PDE inhibitor, and selective inhibitors for various PDE subtypes. Inhibition of PDE subtypes with cilostamide (type III), Ro 20-1724 (type IV) and zardaverine (types III and IV) increased prorenin release. Inhibition of type I (Ca 2+/calmodulin-dependent) PDE by 8-MeoM-IBMX and of type V (cGMP-specific) PDE by zaprinast or dipyridamole did not affect prorenin secretion. The stimulation of prorenin secretion by PDE inhibitors was attenuated by cAMP-dependent protein kinase inhibition. The selective PDE inhibitors caused a parallel increase in media cAMP and prorenin and also increased tissue prorenin levels. These studies demonstrate that cAMP degradation by type III and IV PDE isoenzymes is a major regulatory mechanism for placental prorenin secretion. It is suggested that enhancers of adenylate cyclase activity are constitutively present in placenta and influence prorenin synthesis and release.

First-trimester villous placenta has high prorenin and active renin concentrations

OBJECTIVE: Term villous placental concentrations of prorenin are known to be very low, whereas those of decidua and fetal membranes are high. It has been demonstrated that prorenin synthesis is modulated by hormones in othe reproductive tissues, thus suggesting a means for paracrine regulation in the placenta. This study was performed to test the hypothesis that placental tissue prorenin concentrations may be influenced by gestational age and are temporally related to alterations in the hormonal milieu. STUDY DESIGN: Decidua and villous placental tissue were obtained from term and first-trimester human pregnancies, and concentrations of prorenin, active renin, prolactin, and human chorionic gonadotropin were measured. Values were compared between gestational periods, and relationships between renin and hormone values were analyzed. RESULTS: Prorenin concentrations in first-trimester placenta were nearly 200-fold higher than at term. The proportion of active renin was higher with early gestation. Decidual prorenin and active renin concentrations were similar in both groups. Placental prorenin correlated with chorionic gonadotropin but not prolactin in both groups. CONCLUSIONS: This study demonstrates large differences in placenta prorenin and active renin in villous placental tissue between first-trimester and term gestation tissues, yet there differences were not observed in decidual tissues. The contrast in placental prorenin values observed at the extremes of pregnancy parallel those of placental human chorionic gonadotropin.

Paracrine regulation of the bovine ovarian prorenin-renin-angiotensin-system.

In the bovine ovary, prorenin production by theca cells is known to be regulated by LH. In the present study the aim was to evaluate whether LH-stimulated prorenin production could be further modulated by intraovarian factors in vitro. Theca cells were isolated from bovine ovaries by enzymatic dispersion, purified over Percoll gradient and cultured under serum-free conditions with LH/8Br-cAMP in the absence or presence of different steroids and growth factors and the amount of prorenin secreted into the medium was measured. None of the steroids used (androstendione, estradiol, progesterone) influenced the basal or LH-stimulated prorenin production. In contrast, cytokines and growth factors, like TNF alpha, TGF alpha, TGF beta and bFGF proved to be important regulators of prorenin synthesis. Whereas TNF alpha, TGF alpha and bFGF significantly reduced the LH- and 8Br-cAMP-induced prorenin synthesis at a site distal to cAMP formation, addition of TGF beta led to a further increase in the amount of prorenin secreted into the medium. None of the agonists had an influence on prorenin production by itself. The observed effects of cytokines and growth factors seemed to be confined to prorenin production only, since cell number, cell viability and steroidogenic response were not at all influenced by the agonists. We conclude that, although LH appears to be the primary regulator of ovarian prorenin production, several paracrine/autocrine intraovarian factors may be involved in "finely tuning" the secretion of prorenin, which is necessary for maintaining the differentiated state of the follicle.

Renin processing and secretion in adrenal and retina of transgenic (mREN-2)27 rats

Two extrarenal tissue sources of renin were studied using quantitative assays and immunocytochemical methods during 12 hours following binephrectomy (BNx) in anesthetized hypertensive homozygous Ren-2 transgenic (TG) rats maintained off hypotensive drugs for three weeks. Compared to normal rats, circulating active renin was depressed 50% in conscious TG rats and prorenin was 5- to 10-fold higher. Post-BNx, arterial active and prorenin increased progressively to 10-fold, at which time adrenal venous outputs were 0.1 and 20 mGU/min, respectively. The ratio of active to prorenin (3.1 +/- 0.6%) remained unchanged with increasing plasma levels. Thus, either intrinsic enzyme activity of the transgenic prorenin contributed a constant proportion to the measured active renin, or processing to mature renin was coupled to prorenin synthesis and secretion in extrarenal tissues. In the TG rat eye, renin protein labeling was localized throughout retinal Müller cells with prosequence more obvious posteriorly, consistent with directional processing. Immunogold studies are in progress. In adrenal following BNx, labeling for renin and prosequence increased uniformaly in all zones of the cortex and in scattered medullary chromaffin cells. In cortex, both renin and prosequence were strongly located in intramitochondrial dense bodies. In chromaffin cells, renin labeling was present in both cytoplasmic vesicles and electron-dense granules, while prosequence was predominantly in cytoplasmic vesicles, consistent with processing of prorenin prior to storage in chromaffin granules.

Paracrine regulation of the bovine ovarian prorenin-renin-angiotensin-system

In the bovine ovary, prorenin production by theca cells is known to be regulated by LH. In the present study the aim was to evaluate whether LH-stimulated prorenin production could be further modulated by intraovarian factors in vitro. Theca cells were isolated from bovine ovaries by enzymatic dispersion, purified over Percoll gradient and cultured under serum-free conditions with LH/8Br-cAMP in the absence or presence of different steroids and growth factors and the amount of prorenin secreted into the medium was measured. None of the steroids used (androstendione, estradiol, progesterone) influenced the basal or LH-stimulated prorenin production. In contrast, cytokines and growth factors, like TNFα, TGFα, TGFβ and bFGF proved to be important regulators of prorenin synthesis. Whereas TNFα, TGFα and bFGF significantly reduced the LH-and 8Br-cAMP-induced prorenin synthesis at a site distal to cAMP formation, addition of TGFβ led to a further increase in the amount of prorenin secreted into the medium. None of the agonists had an influence on prorenin production by itself. The observed effects of cytokines and growth factors seemed to be confined to prorenin production only, since cell number, cell viability and steroidogenic response were not at all influenced by the agonists. We conclude that, although LH appears to be the primary regulator of ovarian prorenin production, several paracrine/autocrine intraovarian factors may be involved in “finely tuning” the secretion of prorenin, which is necessary for maintaining the differentiated state of the follicle.

Beta-adrenoceptor activation stimulates, and phosphodiesterase inhibition potentiates, placental prorenin synthesis and release

Beta-adrenoceptor activation stimulates, and phosphodiesterase inhibition potentiates, placental prorenin synthesis and release

Beta-adrenoceptor activation stimulates, and phosphodiesterase inhibition potentiates, placental prorenin synthesis and release.

This study evaluated activation of beta-adrenoceptors and the cAMP pathway on prorenin secretion from human placental explants. For comparative purposes, hCG secretion was also measured. Treatment with selective beta-adrenergic agonists (beta 1-dobutamine and beta 2-terbutaline) produced dose-dependent increases in prorenin secretion, with dobutamine yielding a greater response (10- vs. 6-fold). In contrast, hCG secretion was stimulated only by terbutaline (5-fold). Prorenin and hCG secretory responses were inhibited by corresponding selective receptor antagonists (beta 1-metoprolol and beta 2-ICI 118,551). Selective phosphodiesterase inhibitors were used to evaluate the role of cAMP in mediating these responses. Marked potentiation of beta-adrenoceptor-dependent prorenin secretion was observed with the type III inhibitor, cilostamide (63-76%), and the type IV inhibitor, Ro-201724 (32-43%). Type I (8-methoxymethyl-3-isobutylmethylxanthine) and type V inhibitors (dipyridamole and M&B 22,948) showed no potentiation. These studies demonstrate that activation of both beta 1- and beta 2-receptors stimulates placental prorenin release. The potentiation of beta-adrenergically activated prorenin release by selective inhibitors of phosphodiesterase indicates a coupling of beta-adrenoceptor and adenylate cyclase. The contrast in secretion of prorenin and hCG by selective beta-adrenergic agonists suggests differences in cellular localization. The results indicate that clinically used adrenergic agonists can affect the placental renin-angiotensin system. The role of endogenous activators of beta-adrenoceptors in this system remains to be determined.

Renin processing in cultured juxtaglomerular cells of the hydronephrotic mouse kidney.

We examined renin processing in cultured juxtaglomerular (JG) cells of the hydronephrotic mouse kidney with immunocytochemical and biochemical techniques. Compared with JG cells in normal kidneys, there was less intense labeling for renin protein in mature granules of cultured JG cells. However, pro-renin labeling of transport vesicles and juvenile granules was maintained, suggesting incomplete passage of pro-renin through intermediate and mature granules. Immunogold evidence of exocytosis of mature granules containing renin protein was present at all stages. Labeling of transport vesicles for pro-renin, together with the absence of exocytosis of pro-renin from juvenile granules, indicated that pro-renin was exclusively released by a constitutive process. Active renin release into supernatants decreased with time, whereas the ratio of total renin to active renin increased, indicating that pro-renin synthesis and release were maintained but that the processing of pro-renin to active renin was interrupted. Angiotensin II inhibited and verapamil stimulated active renin release in culture; neither substance affected pro-renin release. Application of secretagogues that act via intracellular calcium or cAMP resulted in depletion of mature granules and their deformation by myelin figures and vacuoles, findings consistent with an exocytosis from mature granules. The absence of effect of any secretagogues on pro-renin release suggests that these stimulatory mechanisms are exclusively post-Golgi. In cultured JG cells in renal explants, renin vesicular transport and granular exocytosis are maintained but a defect in pro-renin passage from juvenile to intermediate granules is apparent.

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

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 proto- and 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.

Cyclic AMP-induced and spontaneous increase in prorenin synthesis and release from human placenta in vitro

Cyclic AMP-induced and spontaneous increase in prorenin synthesis and release from human placenta in vitro

RENIN GENE EXPRESSION IN VARIOUS TISSUES DETERMINED BY SINGLE‐STEP POLYMERASE CHAIN REACTION

1. Renin mRNA is present in the kidney and, in lower concentrations, in many extrarenal tissues and serves as an index of renin gene activity, as well as potential renin or prorenin synthesis in cell populations within those tissues. Unfortunately the quantity can be very low. 2. A new, highly sensitive technique is described for detection of renin mRNA that involves the enzyme Taq polymerase for both reverse transcription of renin mRNA into renin cDNA and for amplification of the 769-1099 nucleotide segment by the polymerase chain reaction (PCR), all of which involves a single reaction mixture. 3. In this way renin mRNA was detected in kidney and several extrarenal tissues as a PCR product of approximately 330 base pairs on agarose gels by ethidium-bromide staining or hybridization probing. The region of renin mRNA chosen for amplification spanned several intron sites in the coding sequence so that the presence of amplicants derived from genomic DNA could be readily discriminated, as a band of approximately 1.5 kilobases. 4. Thus single-step PCR offers a powerful new approach to detection of renin mRNA.