Atrial natriuretic polypeptide inhibits hypertrophy of vascular smooth muscle cells.

Vascular remodeling is central to the pathophysiology of hypertension and atherosclerosis. Recent evidence suggests that vasoconstrictive substances, such as angiotensin II (AII), may function as a vascular smooth muscle growth promoting substance. To explore the role of the counterregulatory hormone, atrial natriuretic polypeptide (ANP) in this process, we examined the effect of ANP (alpha-rat ANP [1-28]) on the growth characteristics of cultured rat aortic smooth muscle (RASM) cells. ANP (10(-7) M) significantly suppressed the proliferative effect of 1% and 5% serum as measured by 3H-thymidine incorporation and cell number, confirming ANP as an antimitogenic factor. In quiescent RASM cells, ANP (10(-7), 10(-6) M) significantly suppressed the basal incorporations of 3H-uridine and leucine by 50 and 30%, respectively. ANP (10(-7), 10(-6) M) also suppressed AII-induced RNA and protein syntheses (by 30-40%) with the concomitant reduction of the cell size. Furthermore, ANP also significantly attenuated the increase of 3H-uridine and leucine incorporations caused by transforming growth factor-beta (4 x 10(-11), 4 x 10(-10) M), a potent hypertrophic factor. These results indicate that ANP possesses an antihypertrophic action on vascular smooth muscle cells. Down-regulation of protein kinase C by 24-h treatment with phorbol 12,13-dibutyrate did not inhibit ANP-induced suppression on 3H-uridine incorporation. Based on the observation that ANP was more potent than a ring-deleted analogue of ANP on inhibiting 3H-uridine incorporation, we conclude that the ANP's inhibitory effect is primarily mediated via the activation of a guanylate cyclase-linked ANP receptor(s). Indeed 8-bromo cGMP mimicked the antihypertrophic action of ANP. Accordingly, we speculate that in addition to its vasorelaxant and natriuretic effects, the antihypertrophic action of ANP observed in the present study may serve as an additional compensatory mechanism of ANP in hypertension.

Induction of platelet-derived growth factor A-chain and c-myc gene expressions by angiotensin II in cultured rat vascular smooth muscle cells.

Recently, angiotensin II (Ang II) has been shown to cause hypertrophy of cultured quiescent rat aortic smooth muscle (RASM) cells. This observation along with the demonstration of angiotensinogen mRNA in the vessel wall has led us to postulate a role for vascular angiotensin in hypertensive blood vessel hypertrophy. To investigate further the possible molecular mechanisms, we examined the effect of Ang II on the expression of two genes known to be involved with cellular growth response. Near-confluent RASM cells were made quiescent by 48-h exposure to a defined serum-free medium. Ang II (10(-6) to 10(-11) M) resulted in an induction of the protooncogene c-myc mRNA within 30 min which persisted for 6 h. Interestingly, 6 h after the addition of Ang II, platelet-derived growth factor (PDGF) A-chain mRNA expression was elevated, peaked in 9 h, and persisted for 11 h. This was accompanied with a 15-20-fold increase in PDGF concentration in the culture medium. These effects were dose-dependent and were blocked by saralasin. Whereas the inhibition of protein synthesis by cycloheximide resulted in a stabilization of c-myc mRNA, cycloheximide abolished the elevation of the PDGF A-chain mRNA. Taken together, our data show that exposure of RASM cells to Ang II results in the sequential activation of c-myc and PDGF A-chain mRNA expressions. This sequential activation of protooncogene and growth factor gene may be an important mechanism in angiotensin-induced smooth muscle growth and hypertrophy.

Vascular smooth muscle cell hypertrophy vs. hyperplasia. Autocrine transforming growth factor-beta 1 expression determines growth response to angiotensin II.

Recent observations in our laboratory suggest that angiotensin II (Ang II) is a bifunctional vascular smooth muscle cell (VSMC) growth modulator capable of inducing hypertrophy or inhibiting mitogen-stimulated DNA synthesis. Because transforming growth factor-beta 1 (TGF beta 1) has similar bifunctional effects on VSMC growth, we hypothesized that autocrine production of TGF beta 1 may mediate the growth modulatory effects of Ang II. Indeed, this study demonstrates that Ang II induces a severalfold increase in TGF beta 1 mRNA levels within 4 h that is dependent on de novo protein synthesis and appears to be mediated by activation of protein kinase C (PKC). Ang II not only stimulates the synthesis of latent TGF beta 1, but also promotes its conversion to the biologically active form as measured by bioassay. The coincubation of VSMCs with Ang II and control IgG has no significant mitogenic effect. However, the co-administration of Ang II and the anti-TGF beta 1 antibody stimulates significantly DNA synthesis and cell proliferation. We conclude that: (a) Ang II induces increased TGF beta 1 gene expression via a PKC dependent pathway involving de novo protein synthesis; (b) Ang II promotes the conversion of latent TGF beta 1 to its biologically active form; (c) Ang II modulates VSMC growth by activating both proliferative and antiproliferative pathways; and (d) Autocrine active TGF beta 1 appears to be an important determinant of VSMC growth by hypertrophy or hyperplasia.

Distinct nuclear proteins competing for an overlapping sequence of cyclic adenosine monophosphate and negative regulatory elements regulate tissue-specific mouse renin gene expression.

The mouse renin locus (Ren-1d) exhibits specific patterns of tissue expression. It is expressed in kidney but not submandibular gland (SMG). This locus contains a negative regulatory element (NRE) and a cAMP responsive element (CRE) that share an overlapping sequence. In the kidney, CRE binding proteins (CREB) and NRE binding proteins (NREB) compete for binding to this sequence, with the CREB having a greater affinity. In the SMG, CREB is inactivated by an inhibitory protein, permitting NREB to bind to the sequence, thus inhibiting Ren-1d expression. We hypothesize that the competition between NREB and CREB for this sequence governs tissue-specific expression of mouse renin. We speculate that this may be a general paradigm that determines tissue-specific gene expression.

H19, a developmentally regulated gene, is reexpressed in rat vascular smooth muscle cells after injury.

Vascular smooth muscle cell migration, proliferation, and differentiation are central to blood vessel development. Since neointimal formation after vascular injury may require the reexpression of a smooth muscle developmental sequence, we examined the expression of H19, a developmentally regulated gene, in rat blood vessels. Expression of the H19 gene is associated with the differentiation process that takes place during development of many tissues. Consistent with this, H19 was highly expressed in the 1-d-old rat aorta but was undetectable in the adult. H19 transcripts were only minimally detected in uninjured carotid artery but were abundant at 7 and 14 d after injury and were localized by in situ hybridization, primarily to the neointima. H19 transcript were undetectable in proliferating neointimal cells in culture but became highly abundant in postconfluent, differentiated neointimal cells. H19 transcripts were only minimally expressed in adult medial smooth muscle cells grown under the identical conditions. Thus, H19 may play an important role in the normal development and differentiation of the blood vessel and in the phenotypic changes of the smooth muscle cells, which are associated with neointimal lesion formation. The vascular injury model may be a useful system to use in examining the function of H19.

Sodium regulation of angiotensinogen mRNA expression in rat kidney cortex and medulla.

Rat liver angiotensinogen cDNA (pRang 3) and mouse renin cDNA (pDD-1D2) were used to identify angiotensinogen and renin mRNA sequences in rat kidney cortex and medulla in rats on high and low salt diet. Angiotensinogen mRNA sequences were present in renal cortex and medulla in apparently equal proportions, whereas renin mRNA sequences were found primarily in renal cortex. Average relative signal of rat liver to whole kidney angiotensinogen mRNA was 100:3. Densitometric analysis of Northern blots demonstrated that renal cortical angiotensinogen mRNA concentrations increased 3.5-fold (P less than 0.001) and medulla, 1.5-fold (P less than 0.005) on low sodium compared with high sodium diet, whereas renal cortex renin mRNA levels increased 6.8-fold (P less than 0.0005). Dietary sodium did not significantly influence liver angiotensinogen mRNA levels. These findings provide evidence for sodium regulation of renal renin and angiotensinogen mRNA expressions, which supports potential existence of an intrarenally regulated RAS and suggest that different factors regulate renal and hepatic angiotensinogen.

Multiple autocrine growth factors modulate vascular smooth muscle cell growth response to angiotensin II.

Angiotensin (Ang) II stimulates hypertrophic growth of vascular smooth muscle cells (VSMC). Accompanying this growth is the induction of the expression of growth-related protooncogenes (c-fos, c-jun, and c-myc), as well as the synthesis of the autocrine growth factors, such as PDGF-A and TGF-beta 1. In this study, we demonstrate further that Ang II also induces the synthesis of basic fibroblast growth factor (bFGF), a potent mitogen for VSMC. To examine how these factors interact to modulate the growth response of VSMC to Ang II, we used antisense oligomers to determine the relative contribution of these three factors. Treatment of confluent, quiescent smooth muscle cells with specific antisense oligomers complementary to bFGF, PDGF-A, and TGF-beta 1 efficiently inhibited the syntheses of these factors. Our results demonstrate that in these VSMC, TGF-beta 1 affects a key antiproliferative action, modulating the mitogenic properties of bFGF. Autocrine PDGF exerts only a minimal effect on DNA synthesis. An imbalance in these signals activated by Ang II may result in abnormal VSMC growth leading to the development of vascular disease.

Localization and differential regulation of angiotensinogen mRNA expression in the vessel wall.

Recent data demonstrate the existence of a vascular renin angiotensin system. In this study we examine the localization of angiotensinogen mRNA in the blood vessel wall of two rat strains, the Wistar and Wistar Kyoto (WKY), as well as the regulation of vascular angiotensinogen mRNA expression by dietary sodium. Northern blot analysis and in situ hybridization histochemistry demonstrate that in both strains angiotensinogen mRNA is detected in the aortic medial smooth muscle layer as well as the periaortic fat. In WKY rats fed a 1.6% sodium diet, angiotensinogen mRNA concentration is 2.6-fold higher in the periaortic fat than in the smooth muscle, as analyzed by quantitative slot blot hybridization. Angiotensinogen mRNA expression in the medial smooth muscle layer is sodium regulated. After 5 d of a low (0.02%) sodium diet, smooth muscle angiotensinogen mRNA levels increase 3.2-fold (P less than 0.005) as compared with the 1.6% sodium diet. In contrast, angiotensinogen mRNA level in the periaortic fat is not influenced by sodium diet. In summary, our data demonstrate regional (smooth muscle vs. periaortic fat) differential regulation of angiotensinogen mRNA levels in the blood vessel wall by sodium. This regional differential regulation by sodium may have important physiological implications.

In vivo identification of a negative regulatory element in the mouse renin gene using direct gene transfer.

DBA/2J mouse contains two renin gene loci (Ren1d and Ren2d). Ren2d but not Ren1d is expressed in submandibular gland (SMG) while both are expressed in the kidney. Based on vitro studies, we have postulated that a negative regulatory element (NRE) in the renin gene promoter is involved in its tissue-specific expression. In this study, we examined the molecular mechanism at the in vivo level using direct gene transfer. Fragments of the Ren1d or Ren2d promoter were fused to a chloramphenicol acetyltransferase (CAT) gene expression vector. These constructs complexed in fusogenic liposomes were injected directly into the mouse SMG or intraarterially into the mouse kidney via the renal artery. The vector containing the CAT exhibited readily detectable in vivo expressions in both SMG and kidney. In the SMG, Ren1d fragment containing the NRE abolished CAT expression while deletion of the NRE restored CAT expression. The homologous fragment from the Ren2d promoter did not inhibit CAT expression while deletion of the 150-bp insertion resulted in the inhibition. Cotransfection of Ren1d construct with Ren1d-NRE oligonucleotides as transcriptional factor decoy restored CAT expression. Contrary to the SMG, transfection with Ren1d fragment-CAT construct or Ren2d fragment-CAT construct into the kidney resulted in similar levels of CAT expression. Interestingly, human c-myc NRE oligonucleotides which share homology with Ren1d-NRE competed effectively with these oligonucleotides for the regulation of Ren1d gene expression in vivo. This NRE sequence is also homologous to silencer elements found in multiple mammalian genes, suggesting the presence of a family of NRE/NRE binding proteins regulating expression of diverse genes.

Autocrine and paracrine effects of atrial natriuretic peptide gene transfer on vascular smooth muscle and endothelial cellular growth.

In addition to the atria, recent evidence suggests that atrial natriuretic peptide (ANP) is also synthesized in other tissues. Of particular interest is the location of ANP mRNA in the vessel wall. We and others have shown that exogenously added ANP inhibited the growth of endothelial cells and vascular smooth muscle cells (VSMC) in culture. However, it is not known if the locally synthesized ANP would act similarly. Because cultured endothelial cells and VSMC have lost the ability to express the endogenous ANP gene, we have transfected cells in culture with an expression vector expressing rat ANP and have examined the effects on growth. Cultured endothelial cells transfected with an ANP expression vector synthesized and secreted high levels of ANP. These cells also showed significantly lower rates of DNA synthesis under basal and fibroblast growth factor (FGF)-stimulated conditions. Addition of conditioned medium from endothelial cells transfected with ANP vector to nontransfected endothelial cells resulted in the significant increase in cyclic GMP. Similarly, conditioned media collected from endothelial cells transfected with ANP vector also decreased DNA synthesis in VSMC. Coculture of ANP-transfected endothelial cells with quiescent VSMC showed that released ANP from endothelial cells inhibited DNA synthesis in VSMC. Finally, we examined the autocrine effect of direct transfection of ANP vector into VSMC. Transfection of the ANP vector decreased DNA synthesis in VSMC under basal and angiotensin II-stimulated conditions. Similarly, transfection of the ANP vector resulted in a decrease in the PDGF and serum (5%)-stimulated DNA synthesis of VSMC. These results demonstrate that endogenously produced ANP can exert autocrine and paracrine inhibitory effects on endothelial cell and VSMC growth. In vivo gene transfer of ANP may provide us with the opportunity of gene therapy for vascular diseases in which the abnormalities are vasoconstriction and pathological growth.

Induction of angiotensin converting enzyme in the neointima after vascular injury. Possible role in restenosis.

Angiotensin II (Ang II) promotes growth of vascular smooth muscle cells in vitro. Consistent with this, Ang II enhances neointimal proliferation in vivo after vascular injury, while angiotensin converting enzyme (ACE) inhibitors attenuate this process. Since tissue ACE plays a key role in the control of local Ang II production, we examined whether vascular injury resulted in an increase in vascular ACE expression that may result in increased Ang II production. Abdominal aorta of Sprague-Dawley rats were injured with a 2 French balloon catheter. Morphometrical changes, ACE enzymatic activity, and localization of ACE by immunohistochemistry in injured and uninjured aorta were analyzed. Vascular ACE activity in the injured aorta was significantly higher than in the uninjured aorta, while serum and lung ACE levels were not different between the two groups. The cellular distribution of the ACE protein in the neointima was similar to that of alpha smooth muscle actin but differed from those of endothelial (von Willebrand factor) or monocytes/macrophages (ED-1) markers, demonstrating that ACE was expressed in neointimal smooth muscle cells. These data demonstrate that vascular injury results in the induction of vascular ACE and suggest that the inhibition of vascular ACE may be important in the prevention of restenosis after balloon injury.

Absence of control of poly(A)(+) messenger RNA translation in growth-stimulated mouse 3T6 fibroblasts.

To better understand the mechanism by which the rate of protein synthesis is regulated in growth-stimulated mouse 3T6 fibroblasts, we have determined the proportion of cytoplasmic poly(A)(+) mRNA that is associated with polysomes. We found that this proportion is nearly the same (70-80%)in resting, serum-stimulatd, or exponentially growing cells. This indicates that the increase in the rate of protein synthesis following serum stimulation of 3T6 cells is regulated primarily by the increase in mRNA content rather than by an increase in the fraction of total mRNA engaged in protein synthesis. We also show that in detergent-lysed 3T6 cells, up to half of the subpolysomal poly(A)(+) mRNA is of mitochondrial origin. This conclusion is based on analysis of the size of the mRNA and its poly(A), its subcellular location and the inhibition of the labeling of this mRNA by ethidium bromide. The physical properties of subpolysomal mRNA of nuclear origin are similar to those of polysomal mRNA.

Sibling-based association study of the PPARgamma2 Pro12Ala polymorphism and metabolic variables in Chinese and Japanese hypertension families: a SAPPHIRe study. Stanford Asian-Pacific Program in Hypertension and Insulin Resistance.

The peroxisome proliferator activated receptor (PPAR) gamma2 is a transcription factor that has been shown to be involved in adipocyte differentiation, adipogenesis, and insulin sensitivity. To address the role of PPARgamma2 in glucose homeostasis and insulin sensitivity, among many other objectives, we conducted a sibling-controlled association study in a multicenter program - the Stanford Asian-Pacific Program in Hypertension and Insulin Resistance (SAPPHIRe). Approximately 2525 subjects in 734 Chinese and Japanese families have been recruited from six field centers for SAPPHIRe. In total, 1702 subjects including parents and siblings from 449 families have been genotyped for PPARgamma2, of which 328 families were Chinese and 121 Japanese. Only 88 subjects of the 1525 siblings screened for the P12A polymorphism were found to be carriers of the A variant, the most common variant of the PPARgamma2 gene. A variant frequencies of the siblings were 4.27% in Chinese and 2.72% in Japanese. A sibling-controlled association study was performed through genetically discordant sibships (i.e., P/P genotype vs. P/A + A/A genotypes). Specifically, we examined whether there were differences in metabolic variables between the discordant siblings within families. In total, 88 subjects carrying either 1 or 2 A alleles had at least one sibling who was discordant for the P12A polymorphism, yielding a total of 180 individuals from 47 families for analyses, among which 92 siblings were homozygous for wild-type P allele. Siblings with the A variant tended to have lower levels of fasting plasma glucose (OG-10), and lower glucose levels at 60 min following oral glucose loading after adjusting for age, gender, and body mass index. Using a mixed model treating family as a random effect, we found that P12A polymorphism of the PPARgamma2 gene contributes significantly to the variance in fasting plasma glucose, glucose level at 60 min, and insulin-resistance homeostasis model assessment. Our results suggest that within families siblings with the A variant in the PPARgamma2 gene may be more likely to have better glucose tolerance and insulin sensitivity independent of obesity in Chinese and Japanese populations.

Intracellular trafficking of angiotensin II and its AT1 and AT2 receptors: evidence for selective sorting of receptor and ligand.

Angiotensin II (Ang II) binds to two different receptor subtypes, AT1 and AT2 receptors. In many cases, receptor stimulation by Ang II is followed by a rapid desensitization of the intracellular signal transduction and a decrease in cell surface receptor number. The present study was designed to examine by immunofluorescence microscopy the cellular trafficking pathways of Ang II and its AT1a and AT2 receptors in human embryonal kidney 293 cells stably expressing these receptor subtypes. Fluorescently labeled Ang II and AT1a receptors were rapidly internalized into endosomes. AT2 receptors were localized in the plasma membrane and did not undergo endocytosis upon agonist stimulation. After removal of agonist, AT1a receptors recycled to the plasma membrane, whereas fluorescently labeled Ang II was targeted to the lysosomal pathway. Even though no further loss of surface receptor was measurable by ligand binding at steady state, fluorescein-Ang II was continuously internalized, and cycling of receptor between endosomal vesicles and the plasma membrane was detected by antibody feeding. These experiments provide evidence for subtype-specific receptor sorting and internalization of Ang II and its AT1a receptor as a receptor-ligand complex, and they suggest that the sequestration of receptors into endosomes is in dynamic equilibrium with receptor cycling to the plasma membrane. Finally, internalization of AT1a receptors and Ang II persists after desensitization mechanisms have attenuated Ca2+ and inositol 1,4,5-trisphosphate signaling.

Modulation of vascular development and injury by angiotensin II.

OBJECTIVE: To examine the exact profile of expression and to determine the functional significance of the angiotensin II (Ang II), type I (AT1) and type 2 (AT2) receptors during rat aortic development and following rat carotid artery balloon injury. METHODS: AT1 and AT2 mRNA levels in rat aortae were measured using a quantitative reverse transcription polymerase chain reaction technique. Ang II receptor function was assessed by quantitating the effects of AT1 (DuP753) and AT2 (PD123319) receptor antagonists during these processes. RESULTS: During aortic development, AT1 expression was detected on gestational day 14, increased until embryonic day 16 (E16), after which, levels were similar throughout postnatal development. Conversely, AT2 mRNA first appeared at E16, reached maximal levels between E19 and neonatal day 1, and decreased thereafter. DNA synthesis rates decreased with aortic development (high at E15, 73.8 +/- 3.1%; dropping to 37.5 +/- 2.3% by E21). Whereas AT1 receptor antagonism accelerated this developmentally regulated decrease in DNA synthesis. AT2 receptor antagonism blunted this decrease. Because activated adult medial smooth muscle cells express a neonatal phenotype after vascular injury, we assessed Ang II receptor levels and function after carotid artery balloon injury. Both receptor subtypes increased; however, AT2 receptor mRNA expression peaked earlier than AT1 (48 to 72 h after injury). As with aortic development, DNA synthesis occurring between 24 to 48 h after injury (when AT2 receptors constitute 10% of the Ang II receptor population) decreased in DuP753-treated animals and increased in PD123319-treated animals. CONCLUSION: These results indicate that Ang II receptors play a role in vascular development by promoting opposing effects on vascular smooth muscle cell growth.

Mechanisms of natriuretic-peptide-induced growth inhibition of vascular smooth muscle cells.

OBJECTIVE: While natriuretic peptides can inhibit growth of vascular muscle cells (VSMC), controversy exists as to whether this effect is mediated via the guanylate cyclase-coupled receptors, NPR-A and NPR-B, or the clearance receptor, NPR-C. The original aim of this study was to examine the mechanism by which the NPR-C receptor regulates growth. METHODS: Rat VSMC were characterized with regard to natriuretic peptide receptor expression by RT/PCR and radioligand binding studies. The effect on growth following addition of the peptides and the ligands for NPR-C was measured by [3H]thymidine incorporation. Cyclic guanosine monophosphate (cGMP) levels were determined by radioimmunoassay and mitogen activating protein kinase activity was based on the phosphorylation of myelin basic protein. RESULTS: In rat VSMC, passages 4-12, both atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) dose-dependently inhibited serum and PDGF-induced VSMC growth. In contrast, NPR-C specific ligands alone had no effect on cell growth but enhanced growth inhibition when co-administered with ANP and CNP. ANP and CNP also decreased PDGF-BB-stimulated MAP kinase activity. Once again, NPR-C specific ligands alone had no effect but enhanced the effects of ANP. Furthermore, a cGMP specific phosphodiesterase inhibitor dose-dependently inhibited VSMC growth and markedly enhanced natriuretic-peptide-induced inhibition at low peptide concentrations. To examine a potential mechanism for the controversy concerning the NPR-C, we investigated the autocrine expression of ANP and CNP by VSMC and found that mRNA encoding both peptides could be detected by RT/PCR. CONCLUSION: Our findings indicate that the guanyl-cyclase-linked receptors mediate the antiproliferative actions of the natriuretic peptides on vascular smooth muscle cell growth. Moreover, we hypothesize that the apparent inhibition of growth by NPR-C specific ligands reported by others may be due to stabilization of natriuretic peptides produced by the cultured VSMC and subsequent action of these peptides at guanyl-cyclase-linked receptors.

A compendium of gene expression in normal human tissues.

This study creates a compendium of gene expression in normal human tissues suitable as a reference for defining basic organ systems biology. Using oligonucleotide microarrays, we analyze 59 samples representing 19 distinct tissue types. Of approximately 7,000 genes analyzed, 451 genes are expressed in all tissue types and designated as housekeeping genes. These genes display significant variation in expression levels among tissues and are sufficient for discerning tissue-specific expression signatures, indicative of fundamental differences in biochemical processes. In addition, subsets of tissue-selective genes are identified that define key biological processes characterizing each organ. This compendium highlights similarities and differences among organ systems and different individuals and also provides a publicly available resource (Human Gene Expression Index, the HuGE Index, http://www.hugeindex.org) for future studies of pathophysiology.

Regulation of extra-renal renin during ontogeny.

Previous studies demonstrated that the level of renin in the male submandibular gland (SMG) of mouse strains that contain renin genes [e.g. Cr1:CD-1(1CR)BR] increased dramatically at puberty, but a less pronounced response was seen in the C57BL/10J (a strain that has a single renin gene). However, the expression of renin in kidney and other extrarenal tissues throughout growth and development has not been examined. In this study we characterized developmental changes in renal renin and certain extrarenal renin levels in the male and female CD-1 mouse and male C-57 mouse. Renal renin activity remains relatively constant throughout ontogeny in male and female CD-1 mice and in the C-57 male mouse. In the CD-1 male mouse, SMG renin levels vary during ontogeny, coincident with periods in growth and development that are associated with hormonal shifts. Glandular renin levels are higher in the neonatal period than at 2-3 weeks of age, and then rise dramatically with the onset of puberty. Renin levels in the adrenal and testis of the CD-1 male mouse follow a similar temporal pattern. However, this dramatic increase in gonadal, adrenal, and SMG renin in the CD-1 male mouse at puberty is not seen in the organs from the CD-1 female mouse or those of the C-57 male mouse. Taken together, the present results demonstrated that the expression of extrarenal sources of renin is under genetic and hormonal influences. In addition, our data suggest that control of extrarenal renin expression may differ from that of renal renin.

Identification of "B" receptor for natriuretic peptide in human kidney.

Three distinct receptor types for natriuretic peptides (NP) have been identified in human tissue. "A" and "B" receptors initiate biological actions, whereas the "C" receptor has a clearance function. It has been proposed that the natural ligand for the B receptor is c-type natriuretic peptide (CNP), rather than atrial natriuretic peptide (ANP), and that the B receptor is only found in the central nervous system (CNS) and is responsible for all NP-mediated effects in the CNS. Contrary to this hypothesis, we have identified, by means of the polymerase chain reaction (PCR), the B receptor in human kidney tissue. To detect A and C receptors, the PCR reaction was performed with primers which yielded predicted 600 and 378 base pair (bp) products, respectively. For the B receptor, 3 different primer sets were used, resulting in the expected 785, 453 and 228 bp fragments. Restriction mapping of the latter two products with Rsa I yielded the expected fragment numbers and sizes, indicating the PCR products were from B receptor mRNA. These results indicate that the human kidney has B as well as A and C receptors. Thus CNP may have a renal as well as a CNS site of action.

A comparative study of the distributions of renin and angiotensinogen messenger ribonucleic acids in rat and mouse tissues.

Previous studies have reported the presence of renin mRNAs in several mouse tissues and angiotensinogen mRNAs in various rat tissues. Clarification as to whether renin and angiotensinogen mRNAs are coexpressed in the same tissues of the same animal species is important for understanding the biology of the tissue renin-angiotensin system. We employed mouse renin cDNA and rat angiotensinogen cDNA to compare tissue distributions of renin and angiotensinogen in RNAs of the rat and mouse. Both cDNA probes readily cross-hybridize with the corresponding mRNA of the other species. Our results demonstrate several patterns of distribution. Renin and angiotensinogen mRNAs are readily detected in kidney and adrenals of both species. In brain and heart, angiotensinogen mRNAs are present in concentrations that far exceed renin mRNA levels in these organs in both species. In mouse and rat livers, angiotensinogen, but not renin, mRNA is demonstrated. In rat testis, only renin mRNA can be detected, whereas in mouse testes both renin and angiotensinogen mRNA are present. In CD-1 male mouse submandibular gland, renin mRNA exists in high concentrations, whereas angiotensinogen mRNA is present in low levels. In contrast, neither renin nor angiotensinogen mRNA could be detected in rat salivary gland. In summary, our study demonstrates the widespread codistribution of renin and angiotensinogen mRNAs in many tissues of both species, allowing for the possibility of local angiotensin production. However, tissue and species differences in these gene expressions also exist. Understanding differential tissue expressions of these genes will provide additional important insight into the biology of the renin-angiotensin system.