Genomic characterization of the sheep vasopressin V1a receptor gene and promoter, with assignment to bands q23-24 of sheep chromosome 3 and cattle chromosome 5.

Arginine vasopressin interacts with the vasopressin type 1a receptor (V1aR) to initiate physiological effects such as vasoconstriction of blood vessels and glycogenolysis. AVP is also involved in central nervous effects such as body homeostasis and blood pressure control. The complete genomic organization of the sheep V1aR gene has been determined, including the presence of one major and two minor transcriptional start sites at -321, -206 and -91bp respectively, relative to the ATG codon. Another more distal minor transcriptional start site was also localized between nucleotides -997 and -892 relative to the ATG codon. One intron exists in the sheep V1aR gene and potential cis- and trans- acting sites were identified in the sheep V1aR promoter. The promoter was also compared to the rat V1aR promoter. The sheep V1aR promoter displays features typical of housekeeping genes, although tissue-specific expression does not support this. V1aR mRNA is absent in the adult sheep liver but not the kidney. One copy of the V1aR gene exists in the sheep genome, which was localized to chromosome 3q23-24, and to the homoeologous position, 5q23-24 in cattle.

Rapid determination of transgene copy number in stably-transfected mammalian cells by competitive PCR.

We describe here an application of the competitive PCR technique to the analysis of copy number of recombinant rat parathyroid hormone-related protein (rPTHrP) gene in stably-transfected murine erythroleukemia (MEL) cell lines. A single-copy reference gene (endogenous mouse PTHrP gene or mPTHrP) is used as an internal control. This control gene, present in the genome of MEL cells, shares the same primer binding sites as the rPTHrP cDNA but contains an internal PvuII site, which allows resolution of the amplified products after restriction enzyme digestion by polyacrylamide gel electrophoresis (PAGE). The transgene copy number is determined by the ratio of band intensity of the rPTHrP product to that of the mPTHrP product. Using this method, we have determined the copy number of the rPTHrP transgene from isolated genomic DNA, and compared the results with those obtained from Southern blot analysis. In addition, we have demonstrated that the procedure can be applied very simply to whole MEL cells without DNA extractions and that as few as 10(4) cells are required for the analysis.

Actions of endothelin-1 on cultured rat renomedullary interstitial cells are modulated by nitric oxide.

1. Cultured renomedullary interstitial cells (RMIC) isolated from 4-week-old Sprague-Dawley rat kidneys possess ETA receptors, as identified by reverse transcription-polymerase chain reaction (RT-PCR). 2. Treatment with endothelin (ET)-1 (10(-6) mol/L) increases the intracellular inositol 1,4,5-trisphosphate concentrations within 10 s and intracellular calcium concentrations after 7 s. 3. Endothelin-1 (10(-7) and 10(-10) mol/L) induced increases in intracellular cAMP concentrations, but only in the presence of N omega-nitro-L-arginine, a nitric oxide synthase (NOS) inhibitor. Addition of ET-1 (10(-10) mol/L) to the RMIC culture led to increases in intracellular cGMP concentrations through activation of NOS. 4. In the presence of ET-1 (10(-7) and 10(-10) mol/L) and during NOS inhibition, RMIC responded with increased cell proliferation and extracellular matrix (ECM) synthesis. These responses were abolished by BQ-123 (10(-6) mol/L), suggesting mediation via the ETA receptor subtype. The proliferative effect of ET-1 was also abolished by atrial natriuretic peptide (10(-6) mol/L). 5. The present study provides evidence that binding of ET-1 to ETA receptors on RMIC activates several intracellular second messenger systems that mediate cell proliferation and ECM synthesis. 6. These results also highlight an important interaction between ET-1 and nitric oxide in the control of RMIC function.

Characterization of a specific antibody to the rat angiotensin II AT1 receptor.

We raised a polyclonal antibody against a decapeptide corresponding to the carboxyl terminus of the rat angiotensin II AT1 receptor. This antibody was demonstrated to be specific for the rat receptor according to a number of approaches. These included (a) the ultrastructural localization of immunogold-labeled receptor on the surfaces of zona glomerulosa cells in the adrenal cortex, (b) the specific labeling of Chinese hamster ovarian (CHO) cells transfected with AT1 receptors, (c) the identification of a specific band on Western blots, (d) the immunocytochemical co-localization of angiotensin receptors on neurons in the lamina terminalis of the brain shown to be responsive to circulating angiotensin II, as shown by the expression of c-fos, and (e) the correlation between the expression of the mRNA of the AT1 receptor and AT1 receptor immunoreactivity.(J Histochem Cytochem 47:507-515, 1999)

Rat renomedullary interstitial cells possess bradykinin B2 receptors in vivo and in vitro.

1. Renomedullary interstitial cells (RMIC), abundant throughout the medulla of the kidney, have been demonstrated to have binding sites for many vasoactive peptides, including atrial natriuretic peptide, endothelin, angiotensin II and bradykinin (BK). These observations would support the hypothesis that interactions between RMIC and vasoactive peptides are important in the regulation of renal function. 2. We aimed to localize the BK B2 receptor binding site to RMIC in vivo and to also demonstrate that these receptors are biologically active in vitro. 3. The present study demonstrates BK B2 binding sites on RMIC of the inner stripe of the outer medulla and the inner medulla of the rat kidney in vivo. 4. We further demonstrate that the BK B2 radioligand [125I]-HPP-Hoe140 specifically bound to rat RMIC in vitro. In addition, reverse transcription-polymerase chain reaction detected the mRNA for the BK B2 receptor subtype in cell extracts. 5. For RMIC in vitro, cAMP levels were increased at 1 min and cGMP levels were increased at 2 min after treatment with 10(-10) and 10(-7) mol/L BK, respectively. Inositol 1,4,5-trisphosphate was increased at 10 s treatment with both 10(-6) and 10(-7) mol/L BK. 6. For RMIC in vitro, BK induced an increase in cell proliferation ([3H]-thymidine incorporation) and an increase in extracellular matrix synthesis (ECM; trans-[35S] incorporation), both effects mediated by BK B2 receptors. 7. We conclude that BK B2 receptors are present on RMIC both in vivo and in vitro. These receptors are coupled to intracellular second messenger systems and, in vitro, their stimulation results in cellular proliferation and synthesis of ECM.

Angiotensin receptors in the nervous system.

In addition to its traditional role as a circulating hormone, angiotensin is also involved in local functions through the activity of tissue renin-angiotensin systems that occur in many organs, including the brain. In the brain, both systemic and presumptive neurally derived angiotensin and angiotensin metabolites act through specific receptors to modulate many functions. This review examines the distribution of these specific angiotensin receptors and discusses evidence regarding the function of angiotensin peptides in various brain regions. Angiotensin AT1 and AT2 receptors occur in characteristic distributions that are highly correlated with the distribution of angiotensin-like immunoreactivity in nerve terminals. Acting through the AT1 receptor in the brain, angiotensin has effects on fluid and electrolyte homeostasis, neuroendocrine systems, autonomic pathways regulating cardiovascular function and behavior. Angiotensin AT1 receptors are also found in many afferent and efferent components of the peripheral autonomic nervous system. The role of the AT2 receptor in the brain is less well understood, although recent knockout studies point to an involvement with behavioral and cardiovascular functions. In addition to the AT1 and AT2 receptors, receptors for other fragments of angiotensin have been proposed. The AT4 binding site, which binds angiotensin, has a widespread distribution in the brain quite distinct from that of the AT1 and AT2 receptors. It is associated with many cholinergic neuronal groups and also several sensory nuclei, but its function remains to be determined. Our discovery that another brain-derived peptide binds to the AT4 binding site in the brain and may represent the native ligand is discussed. Overall, the distribution of angiotensin receptors in the brain indicate that they play diverse and important physiological roles in the nervous system.

Angiotensin II inhibits growth of cultured embryonic renomedullary interstitial cells through the AT2 receptor.

The high abundance of angiotensin II (Ang II) AT2, relative to the AT1 receptor subtype in developing kidneys may be related to their potential as mediators of cell growth, although little evidence exists to support this concept. Renomedullary interstitial cells (RMICs) differentiate early in embryonic kidneys and are important in subsequent nephron development. These cells have been shown in vivo to possess AT2 binding sites, although the functional significance of these sites remains unknown. The aim of the current investigation was to examine the actions of Ang II on cultured embryonic renomedullary interstitial cells (ERMICs). 125I-[Sar1, Ile8]Ang II specifically bound to AT1 and AT2 receptors on ERMICs, and their mRNAs were detected by reverse transcription--polymerase chain reaction (RT-PCR). Angiotensin II (10(-6) M) increased intracellular IP3 concentrations at 20 seconds, and decreased intracellular cAMP concentrations after 10 minutes. Angiotensin II (10(-6) M) induced an increase in [3H]thymidine incorporation, mediated through the AT1 receptor subtype. Basic fibroblast growth factor (bFGF; 20 ng/ml) also increased 3[H]thymidine incorporation after 24 hours of treatment, an effect that was attenuated by subsequent addition of Ang II (10(-6) M). This antiproliferative action of Ang II was blocked by PD 123319 (10(-6) M), an AT2 receptor antagonist, and was not affected by losartan (10(-6) M), an AT1 receptor antagonist. These results indicate a dual role for Ang II in regulating ERMIC mitogenesis: a growth stimulating effect mediated by the AT1 receptor subtype, and an antiproliferative effect mediated by the AT2 receptor subtype.

Effects of angiotensin II on cultured rat renomedullary interstitial cells are mediated by AT1A receptors.

Renomedullary interstitial cells (RMICs) are prominent in the inner medullary interstitium and have binding sites for several vasoactive agents, including angiotensin II (ANG II). Although the functional role of RMICs remains largely unknown, it is likely that the interaction between RMICs and vasoactive peptides is important in the regulation of renal function. The current investigation characterizes the cellular responses following treatment of RMICs with ANG II. Studies were performed on RMICs isolated from Sprague-Dawley rat kidneys. 125I-labeled [Sar1,Ile8]ANG II specifically bound to RMICs at sites determined by reverse transcription-polymerase chain reaction to be of the AT1A subtype. ANG II (10(-6) and 10(-10) M) had no effect on either basal or forskolin-stimulated adenosine 3',5'-cyclic monophosphate accumulation in RMICs but increased intracellular inositol 1,4,5-trisphosphate concentration after 10 s and intracellular calcium concentration after 18 s. For RMICs plated at low densities, ANG II (10(-6) M) induced an increase in [3H]thymidine incorporation, mediated through the AT1-receptor subtype. For RMICs plated at high densities, ANG II (10(-6) M) induced an increase in extracellular matrix synthesis as detected by trans-35S incorporation, an effect also mediated by AT1 receptors. We conclude that ANG II AT1A receptors on cultured RMICs are coupled to intracellular second messenger pathways leading to hyperplasia and synthesis of extracellular matrix.

Distribution of AT4 receptors in the Macaca fascicularis brain.

Angiotensin IV (Val Tyr Ile His Pro Phe), administered centrally, increases memory retrieval and induces c-fos expression in the hippocampus and piriform cortex. Angiotensin IV binds to a high affinity site that is quite distinct in pharmacology and distribution from the angiotensin II AT1 and AT2 receptors and is known as the AT4 receptor. These observations suggest that the AT4 receptor may have multiple central effects. The present study uses in vitro receptor autoradiography, and employs [125I]angiotensin IV to map AT4 receptors in the macaca fascicularis brain. The distribution of the AT4 receptor is remarkable in that its distribution extends throughout several neural systems. Most striking is its localization in motor nuclei and motor associated regions. These include the ventral horn spinal motor neurons, all cranial motor nuclei including the oculomotor, abducens, facial and hypoglossal nuclei, and the dorsal motor nucleus of the vagus. Receptors are also present in the vestibular, reticular and inferior olivary nuclei, the granular layer of the cerebellum, and the Betz cells of the motor cortex. Moderate AT4 receptor density is seen in all cerebellar nuclei, ventral thalamic nuclei and the substantia nigra pars compacta, with lower receptor density observed in the caudate nucleus and putamen. Abundant AT4 receptors are also found in areas associated with cholinergic nuclei and their projections, including the nucleus basalis of Meynert, ventral limb of the diagonal band and the hippocampus, somatic motor nuclei and autonomic preganglionic motor nuclei. AT4 receptors are also observed in sensory regions, with moderate levels in spinal trigeminal, gracile, cuneate and thalamic ventral posterior nuclei, and the somatosensory cortex. The abundance of the AT4 receptor in motor and cholinergic neurons, and to a lesser extent, in sensory neurons, suggests multiple roles for the AT4 receptor in the primate brain.

Localization and quantitation of angiotensin AT1 and AT2 receptors in the pregnant and non-pregnant sheep uterus.

The two angiotensin II receptor subtypes, AT1 and AT2, have been reported to be differentially expressed in the myometrial membrane preparations of nulliparous and pregnant sheep, however, their distribution in the sheep reproductive tract has not been reported. The aim of this study is to map the distribution of AT1 and AT2 receptors in the anoestrus reproductive tract of the sheep by quantitative in vitro autoradiography and to investigate if the density and distribution of the receptors change during pregnancy. The AT2 receptor is abundant in a discrete layer in the myometrium of the anoestrus sheep uterus, whilst the AT1 receptor is expressed at lower levels, predominantly in the endometrium. Near-term pregnant uteri, show a marked change in the expression of angiotensin II receptors: the myometrium no longer expresses detectable AT2 receptors but rather, expresses low levels of AT1 receptors. Angiotensin converting enzyme is found in high concentrations in the blood vessels of the pregnant and non-pregnant sheep reproductive system and on the epithelial cells of the fallopian tubes of the non-pregnant sheep. These studies reveal marked reciprocal changes of angiotensin II receptors, with myometrial AT1 receptors increasing during pregnancy, whilst AT2 receptors fall markedly. These changes suggest that angiotensin II may be involved in regulating changes of uterine structure and function during pregnancy by interaction with multiple receptor subtypes.

Angiotensin-converting enzyme (ACE) gene polymorphism in type II diabetic patients with increased albumin excretion rate.

Approximately one in three patients with diabetes is at risk of developing kidney disease, despite current methods of treatment. It has long been suspected that diabetic kidney disease has a genetic basis, but this has been difficult to prove. Polymorphisms of the angiotensin-converting enzyme (ACE) gene have been shown to be related to the occurrence of nephropathy in type I diabetic patients. This study showed that there was no association in the ACE genotype frequency and increased albumin excretion rate in type II diabetic patients.

Sodium status affects GC-B natriuretic peptide receptor mRNA levels, but not GC-A or C receptor mRNA levels, in the sheep kidney.

1. In humans and experimental animals the natriuresis and diuresis resulting from infusion of atrial natriuretic peptide varies with the sodium status of the subject. Tissue binding studies have suggested that this may be related to changes in the renal receptors for the hormone. 2. In order to establish whether these changes are under transcriptional control, we examined the levels of mRNA for the three natriuretic peptide receptors [GC-A, GC-B and clearance (C) receptors] in renal cortex and medulla from six sodium-loaded, six sodium-depleted and four control sheep. cDNA probes specific to each receptor were generated using the polymerase chain reaction. 3. GC-B receptor mRNA levels were increased approximately two-fold in the renal cortex of sodium-depleted animals, whereas there was no influence on GC-B receptor mRNA levels in the renal medulla. There was no significant difference in mRNA levels for the GC-A and C receptors. 4. At present the role of the GC-B receptor and its natural ligand C-type natriuretic peptide in the control of renal function is unknown. The present experiments imply some intrarenal function for the GC-B receptor and its natural ligand, although the site of any such function, e.g. renal vasculature or tubules, remains unclear. In addition, we have shown that if GC-A and C receptor levels in the sheep are modulated by sodium, the regulation occurs beyond the level of gene transcription.

Distribution of angiotensin II receptor subtypes in the rabbit brain.

We have determined the distribution of angiotensin II receptor subtypes in rabbit brain using in vitro autoradiography. AT1 receptors were found in very high concentrations in the forebrain circumventricular organs--the subfornical organ, organum vasculosum of the lamina terminalis, and the median eminence as observed in other mammals. However, there was very little labeling in the area postrema. In the paraventricular nucleus, median preoptic nucleus, supraoptic nucleus there were high levels of predominantly AT1 receptors. High densities of AT1 receptors were also found in the nucleus of the solitary tract and the rostral and caudal ventrolateral medulla. All of these regions have putative roles in the regulation of blood pressure and fluid and electrolyte balance. In the rabbit brain there is less AT2 receptor binding than the rat, with most AT2 binding found in the molecular layer of the cerebellum and in the septohypothalamic nucleus. In the subthalamic nucleus, the mediodorsal and ventroposterior nuclei of the thalamus, locus coeruleus and inferior olivary nuclei, areas containing mostly AT2 receptors in the rat, no binding was detected in the rabbit except in the locus coeruleus which contains moderate levels of AT1 receptors. Taken in conjunction with our previous results in the rat and human brains, these results reveal that AT1 receptors predominate in rostral forebrain, hypothalamus and autonomic control centers of the medulla oblongata in all three species. However, the distribution and density of AT2 bearing sites in regions such as the septum, thalamus subthalamic nuclei, locus coeruleus, cerebellum and inferior olivary nuclei show marked species differences.

The sequence and tissue expression of ovine renin.

The primary structure of the sheep renin precursor has been determined from its cDNA sequence. A library of cDNA clones was constructed from adrenalectomized sheep kidney poly(A)+ RNA and screened for sheep renin sequences with a cloned mouse renin cDNA probe. Of the 300,000 clones generated, 24 were hybridization positive and the nucleotide sequences of two of the longest clones were determined. These clones coded for the mature sheep renin protein and the 3'-untranslated sequence but did not extend to the amino-terminal region of preprorenin. Clones corresponding to the 5' region of renin mRNA were generated by the polymerase chain reaction and their nucleotide sequences determined. The sheep renin precursor consists of 400 amino acids with a putative leader sequence of 14 amino acids and a putative 45 or 53 amino acid prosegment. The mature sheep renin protein has a 73% sequence identity with human renin. Northern analysis demonstrated the presence of renin mRNA in the kidney but not in other tissues in the sheep. While sodium depletion of sheep caused a rise in renin mRNA in the kidney, adrenalectomy also led to a large increase in renal renin mRNA. Southern analysis of genomic DNA suggests that there is only one gene coding for renin in the sheep.

Differential expression of inhibin alpha and beta A subunit genes in rat and mouse ovarian follicles during pregnancy.

Relative levels of rat ovarian alpha inhibin (alpha I) and beta A inhibin (beta AI) mRNAs were measured during pregnancy by dot-blot hybridization of ovarian poly(A+) RNA. Follicular patterns of alpha I and beta AI expression in contralateral ovaries from the same rats were also studied by hybridization histochemistry. Oligodeoxynucleotide probes specific for porcine alpha I and beta AI were synthesized, 32P end-labelled and used as hybridization probes on dot-blots of ovarian RNA and frozen sections of ovarian tissue from pregnant rats. During pregnancy, levels of alpha I and beta AI mRNAs remained fairly constant from day 7 after mating until parturition and then fell within 16 h post partum. In all ovaries observed, expression of inhibin genes was located in granulosa cells of healthy antral follicles. In general, the strongest signals for alpha I and beta AI mRNAs were obtained in large follicles, with weaker signals in smaller follicles. Follicular patterns of alpha I and beta AI expression during pregnancy were often dissimilar when alpha I and beta AI were compared over a range of follicles. Considerable alpha I mRNA was detectable in some follicles in which beta AI was reduced or undetectable, despite strong signals for both alpha I and beta AI in an adjacent follicle. Essentially, alpha I mRNA levels were relatively consistent between groups of follicles, whereas beta AI levels varied considerably. beta AI mRNA was never observed in a follicle in the absence of alpha I mRNA, indicating that activin production in any follicle occurs in the presence of alpha I mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of synthetic oligonucleotide and recombinant DNA probes to study renin gene expression.

Using hybridization histochemistry renin gene expression has been localized in the juxtaglomerular apparatus (JGA) of the renal cortex in both mouse and sheep kidney. This technique also located renin gene expression in afferent arterioles and interlobular arteries distant from the glomerular tuft in lamb renal cortex. A short (30 mer) synthetic oligonucleotide probe, complementary to a region of the mouse submaxillary gland renin gene, specifically labelled mouse submaxillary gland and kidney. Hybridization histochemistry and Northern blot analysis using both the synthetic oligonucleotide (mouse) probe and a 700 base pair recombinant (sheep) probe showed differences in renin gene expression in the kidney in response to Na restriction in the mouse and Na depletion in the sheep.