Increased cyclic guanosine monophosphate production and overexpression of atrial natriuretic peptide A-receptor mRNA in spontaneously hypertensive rats.

Atrial natriuretic peptide (ANP) specifically stimulates particulate guanylate cyclase, and cyclic guanosine monophosphate (cGMP) has been recognized as its second messenger. Spontaneously hypertensive rats (SHR) have elevated plasma ANP levels, but manifest an exaggerated natriuretic and diuretic response to exogenous ANP when compared to normotensive strains. In isolated glomeruli, the maximal cGMP response to ANP corresponds to a 12- to 14-fold increase over basal levels in normotensive strains (Wistar 13 +/- 2; Wistar-Kyoto 12 +/- 2; Sprague-Dawley 14 +/- 2) while a maximal 33 +/- 3-fold elevation occurs in SHR (P < 0.001). This hyperresponsiveness of cGMP is reproducible in intact glomeruli from SHR from various commercial sources. Furthermore, this abnormality develops early in life, even before hypertension is clearly established, and persists despite pharmacological modulation of blood pressure, indicating that it is a primary event in hypertension. In vitro studies have revealed a higher particulate guanylate cyclase activity in membranes from glomeruli and other tissues from SHR. This increase is not accounted for by different patterns of ANP binding to its receptor subtypes between normotensive and hypertensive strains, as assessed by competitive displacement with C-ANP102-121, an analog which selectively binds to one ANP receptor subtype. The hyperactivity of particulate guanylate cyclase in SHR and its behavior under basal, ligand (ANP), and detergent-enhanced conditions could be attributed either to increased expression or augmented sensitivity of the enzyme. Radiation-inactivation analysis does not evoke a disturbance in the size of regulatory elements normally repressing enzymatic activity, while the expression of particulate guanylate cyclase gene using mutated standard of A- and B-receptors partial cDNAs, quantified by polymerase chain reaction (PCR) transcript titration assay, manifests a selective increase of one guanylate cyclase subtype. Our data suggest that in hypertension, genetic overexpression of the ANP A-receptor subtype is related to the exaggerated biological response to ANP in this disease.

Sequence of the cDNA encoding porcine pro-opiomelanocortin.

Messenger RNA was extracted from porcine pituitary pars intermedia and cloned as cDNA by standard methods. The nucleotide sequence encoding porcine pro-opiomelanocortin was established from analysis of two separate cDNA segments having an overlap of 420 bases. The amino acid sequence for the porcine pro-opiomelanocortin protein, which was inferred from the cDNA sequence, was found to correspond exactly to the sequence determined by direct amino acid analysis of the component peptides of pro-opiomelanocortin; namely, the porcine hormones ACTH, beta-lipotropin, gamma-MSH and the connecting peptide. We thus find no evidence for the existence of two porcine pro-opiomelanocortin genes that differ in their coding sequences, as was suggested by the in vitro protein synthesis results of others using mRNA obtained from porcine pituitaries.

The yeast KEX-2-processing endoprotease is active in the Golgi apparatus of transfected NIH 3T3 fibroblasts.

Proteolytic processing of polyprotein precursors at pairs of basic amino acids is a prerequisite for the generation of bioactive peptide hormones. While the mammalian endoproteases responsible for these cleavages are yet to be identified, this function has been unequivocally assigned in yeast to the product of the KEX-2 gene. To study the molecular mechanisms involved in polyprotein processing, we have transfected the yeast KEX-2 gene into mouse NIH 3T3 fibroblasts and established a new cell line (called 2N-DK) where the KEX-2 endoprotease is permanently expressed. Immunofluorescence studies show that the KEX-2 enzyme is retained within the Golgi of the 2N-DK cells. The evidence for this cellular location is supported by measurement of intracellular and extracellular KEX-2 enzyme activity. In this permanently transfected cell line, KEX-2 activity is exclusively intracellular, in contrast to the situation previously described in transiently infected cell lines, where extracellular KEX-2 activity was detected. Furthermore, infection of 2N-DK cells with a recombinant retrovirus expressing a cDNA coding for porcine proopiomelanocortin (POMC) resulted in the synthesis of POMC and its efficient processing into beta-lipotropin and beta-endorphin, two of its physiologically authentic maturation products. These results suggest that in the fibroblast cell line 2N-DK, proteolytic processing of POMC by KEX-2 endoprotease occurs in the Golgi apparatus.

Is atrial natriuretic peptide synthesized and internalized by gonadotrophs?

The present study was designed to determine whether atrial natriuretic peptide (ANP) could be both synthesized and internalized by rat anterior pituitary gonadotrophs. ANP synthesis was assessed by in situ hybridization of ultrathin frozen sections of anterior pituitary to a biotinylated 30-base oligonucleotide to rat ANP mRNA. As revealed by the immunogold technique, only gonadotrophs were labeled by the probe. At the subcellular level, ANP mRNA was observed at both the nuclear and cytoplasmic levels in gonadotrophs, and labeling of the latter compartment was quantitatively more intense. Internalization of ANP was investigated by an in vivo ultrastructural autoradiographic approach. Intravenous injection of [125I]ANP resulted in rapid labeling within 1 min of the plasma membrane, cytoplasmic matrix, secretory vesicle, and mitochondrial compartments and the Golgi apparatus; these compartments were labeled throughout the remainder of the time course studied (1-30 min). Peak labeling of the plasma membrane compartment was at 1 min and diminished from that point; labeling in the Golgi apparatus peaked 5 min postinjection, while in the other compartments labeling was fairly uniform over the time course. The lysosomal compartment was also radiolabeled; however, only 2 and 5 min after injection of [125I]ANP. The findings demonstrate that gonadotrophs can both synthesize and internalize extracellular ANP. These observations can be extended to suggest that ANP has both autocrine and paracrine actions in the anterior pituitary gland. Since the peptide neither stimulates nor antagonizes the release of any anterior pituitary hormone, these actions are probably unrelated to the adenohypophyseal secretory function.

Synthesis, internalization, and localization of atrial natriuretic peptide in rat adrenal medulla.

Some, though not all studies, have indicated that atrial natriuretic peptide (ANP) can bind to adrenal medullary cells. ANP-like immunoreactivity (ANP-LI) has also been identified in catecholamine-secreting cells. Together, these findings suggest that ANP may be taken up and/or synthesized in the adrenal medulla. The present study was designed to ascertain, by in situ hybridization, whether adrenal chromaffin cells could synthesize ANP, to define by an in vivo ultrastructural autoradiographic approach, whether ANP could, in fact, bind to rat adrenal medulla cells, to determine whether there was a cellular [noradrenaline (NA) vs. adrenaline (A)] selectivity in the binding process, and to establish whether extracellular [125I]ANP could be internalized by these cells. The cellular and subcellular distribution of endogenous ANP-LI was also investigated in both cell types by cryoultramicrotomy and immunocytochemical approaches. The in situ hybridization studies indicate the presence of mRNA to ANP in about 15% of adrenal medullary cells. Intravenous injection of [125I]ANP resulted in a 3-fold, preferential and specific radiolabeling of A-as compared to NA-containing cells. In A-containing cells, plasma membranes were significantly labeled 2 and 5 min post injection; cytoplasmic matrix, mitochondria, and secretory granules throughout the time course studied (1-30 min post injection). Lysosomes, rough endoplasmic reticulum, Golgi apparatus, and nuclei were not labeled. ANP-LI was identified in both NA- and A-containing cells; in the former, it was almost exclusively localized in secretory vesicles, in the latter it was detected in plasma membranes, cytoplasmic matrix, nuclear euchromatin, some mitochondria and relatively fewer granules than in NA-containing cells. The findings suggest that ANP may be synthesized primarily in NA-containing cells and that A-containing cells primarily bind and internalize the extracellular (endogenous or exogenous) atrial peptide. The data suggest that ANP secreted by adrenal medullary chromaffin cells may have distal paracrine actions or interactions with coreleased catecholamines and neuropeptides. Binding and internalization may reflect an action of ANP on the secretory function of A-containing cells.

Regulation of natriuretic peptide receptor A and B expression by transforming growth factor-beta 1 in cultured aortic smooth muscle cells.

Two types of natriuretic peptide receptors (NPR-A and NPR-B) are membrane guanylate cyclases whose relative expression varies in different tissues. Because natriuretic peptides have been shown to inhibit aortic smooth muscle proliferation, we investigated the regulation of NPR-A and NPR-B in these cells under different proliferative conditions. NPR subtype mRNA levels were measured by our newly developed quantitative reverse transcription-polymerase chain reaction assay using mutated NPR-A and NPR-B cRNA as internal standards. The functional impact of their expression was determined by atrial natriuretic peptide (ANP)- and C-type natriuretic peptide (CNP)-induced stimulation of cyclic GMP production. In the intact aorta, NPR-B mRNA levels were found to be 10-fold higher than those of NPR-A. This dominance was further amplified (1000-fold) in long-term cultures (10 to 15 passages) of aortic smooth muscle cells (ASMC). Higher cyclic GMP production with CNP than with ANP was observed in cultured ASMC from Wistar-Kyoto (WKY) rats. Similar stimulation by the two agonists was noted in spontaneously hypertensive rat (SHR) cells, paralleled by a 10-fold increase in NPR-A mRNA levels and ANP stimulation of cyclic GMP in hypertensive cells. The present study also evaluated NPR-A and NPR-B mRNA control by transforming growth factor-beta 1 (TGF-beta 1), an important regulator of cell proliferation that is overexpressed in SHR ASMC. TGF-beta 1 decreased both NPR-A and NPR-B mRNA levels with a predominant effect in SHR cells at high cell density.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoptosis in target organs of hypertension.

Apoptosis or programmed cell death frequently parallels abnormalities in cell proliferation and differentiation. As hypertrophy/hyperplasia or remodeling occurs in organs affected by hypertension, we evaluated the degree of apoptosis in the heart, kidney, and brain in situ in genetically hypertensive mice and rats as well as in cultured vascular smooth muscle cells. Apoptosis was characterized by morphological features, DNA fragmentation, and laddering as well as by terminal deoxynucleotidyl transferase labeling of the 3' OH ends of both extracted DNA and tissue sections. The present report provides the first evidence of increased apoptosis in whole organs of genetically hypertensive rat and mouse strains: in the heart of spontaneously hypertensive rats (SHR) and in the heart (ventricular cardiomyocytes), kidney (inner cortex and medulla), and brain (cortex, striatum, hippocampus, and thalamus) of spontaneously hypertensive mice, with a higher effect of apoptotic inducers in cultured aortic smooth muscle cells derived from SHR. Both types of known apoptotic processes, oligonucleosomal cleavage and large DNA fragmentation, were observed in vascular smooth muscle cells, but only the former appeared to be increased in SHR. This study underlines the importance of cell death dysregulation in hypertension, reveals a new route for investigation of the pathogenesis of hypertension, and suggests novel targets of therapeutic intervention.

Construction of cloning vectors using the Vibrio harveyi luminescence genes luxA and luxB as markers.

A synthetic oligodeoxyribonucleotide harboring four new restriction sites was inserted into the luxB gene of Vibrio harveyi. This insertion did not disrupt the reading frame. An active beta-subunit was synthesized since a plasmid with both the luxA and mutated luxB genes conferred upon Escherichia coli the bacterial luciferase (Lux) phenotype in the presence of an aldehyde. Ligation of a piece of foreign DNA at these new cloning sites in the vector extinguish the Lux phenotype of the transformed bacteria. Therefore, the plasmid was used as a cloning vector, and recombinant DNA-containing bacteria were detected by the loss of bioluminescence. To create more versatile plasmids, the intergenic region of phage f1 was inserted outside of the lux genes. The selection by loss of bioluminescence presents several advantages over the white/blue selection of the lacZ gene on indicator plates.

Expression and pharmacological characterization of the human M1 muscarinic receptor in Saccharomyces cerevisiae.

The yeast S. cerevisiae has been examined as a heterologous host for the expression of mammalian neurotransmitter receptors which couple to guanine nucleotide regulatory (G) proteins. A cloned gene encoding the M1 subtype of human muscarinic receptor (HM1) was transformed into S. cerevisiae on a high copy plasmid under the control of the promoter for the yeast alcohol dehydrogenase (ADH) gene. Northern blotting demonstrated the presence of HM1 transcripts in transformants, and crude membranes prepared from these cells showed saturable binding of the muscarinic antagonist [3H]N-methyl scopolamine with a Kd of 179 pM and Bmax of 20 fmol/mg protein. Competition binding studies revealed pharmacological properties for these sites which were comparable to those reported for the M1 site in mammalian tissues. Yeast expressing HM1 did not exhibit high affinity agonist binding or cell-cycle arrest in the presence of muscarinic agonists, indicating that the mammalian receptor did not couple to the endogenous yeast G protein.

Rat granulosa cells express the gonadotrophin-releasing hormone gene: evidence from in-situ hybridization histochemistry.

There is still debate as to whether natural sequence gonadotrophin-releasing hormone (GnRH) is produced in the mammalian gonads and concerning its potential role as a paracrine modulator of gonadal function. To address this question, we have used in-situ hybridization histochemistry with an oligonucleotide probe complementary to the GnRH decapeptide coding sequence, to determine the cellular site(s) of expression of the GnRH gene in rodent ovaries. GnRH mRNA was detected in granulosa and thecal cells from ovarian follicles at all stages of development (primary-->Graafian), with no significant change in grain density during follicular development. The granulosa cell compartment always contained more mRNA than the thecal cell compartment. Corpora lutea expressed the GnRH gene to the same extent as thecal cells. These results indicate that preproGnRH mRNA is detectable under physiological conditions in the mammalian ovary, though whether this produces authentic GnRH decapeptide or an alternative protein product is not known. The physiological significance of these findings remains to be determined.

Sibling resemblance of erythrocyte ion transporters in French-Canadian sibling-pairs affected with essential hypertension.

OBJECTIVES: Erythrocyte Na+/Li+ countertransport and Na+,K+ cotransport are increased in some Caucasians with essential hypertension. This study examines the relative contributions of genetic and shared environmental factors to the activity of these ion carriers in French-Canadian sibling-pairs affected with essential hypertension. DESIGN: The activity of Na+/Li+ countertransport and Na+,K+ cotransport (rate of Na+ o-dependent Li+ efflux and bumetanide-sensitive 86Rb influx, respectively) was measured in 122 French-Canadian siblings with essential hypertension, including 36 brother/brother and 48 sister/sister pairs. Sibling/sibling correlations were estimated using the FCOR program of the S.A.G.E. package. RESULTS: Na+/Li+ countertransport and Na+,K+ cotransport were respectively higher by 27% (P = 0.002) and 42% (P = 0.0009) in erythrocytes from men compared with women. Intra-individual correlation analysis did not reveal a significant effect of age on the activity of these ion transporters in both males and females, and an influence of plasma lipids (triglycerides, cholesterol, low-density lipoprotein, high-density lipoprotein) in females. In males, Na+,K+ cotransport was correlated with the level of serum triglycerides only (P = 0.01). Familial correlation analysis showed that sibling resemblance of Na+/Li+ countertransport and Na+,K+ cotransport was higher in men (r = 0.26 and 0.39) than in women (r = 0.01 and 0.03, respectively). CONCLUSION: The present data indicate that different factors contribute to the regulation of monovalent ion carriers in erythrocytes from Caucasian men and women with essential hypertension. The activity of erythrocyte Na+/Li+ countertransport and Na+,K+ cotransport appears to be more strongly determined by inheritable factors in men than in women.

Ultrastructural non-radioactive in situ hybridization of GH mRNA in rat pituitary gland: pre-embedding vs ultra-thin frozen sections vs post-embedding.

In situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultra-thin frozen sections, or on ultra-thin sections of tissues embedded in hydrophilic resin such as Lowicryl. With the purpose of comparing the sensitivity, resolution, and ultrastructural preservation of these three methods, we examined the expression of the growth hormone (GH) gene in anterior pituitary cells by in situ hybridization at the ultrastructural level, using a synthetic oligonucleotide complementary to the codons of the mRNA from Gln 45 to Ser 54 labeled at the 3' end of biotin-21dUTP. All these methods gave similar results: mRNA was located on the lamellar endoplasmic reticulum of somatotrophs. The pre-embedding method gave the best ultrastructural preservation, with low resolution with the enzymatic detection system and an intermediate sensitivity. A probe concentration of 10 pmol/ml was sufficient to obtain a signal. With this method gold particles could not be used without pre-treatment. The frozen section method gave the best sensitivity (a signal was observed with 4 pmol/ml of probe) but the lowest ultrastructural preservation. On ultra-thin Lowicryl sections, resolution was as high as with the frozen-section method, ultrastructural conservation was intermediate, and sensitivity was low. These results indicate that the last method seems to be a good compromise between sensitivity and ultrastructural preservation.

Ultrastructural distribution of growth hormone (GH) mRNA and GH intron I sequences in rat pituitary gland: effects of GH releasing factor and somatostatin.

We have measured the distribution of growth hormone (GH) mRNA or intron I sequences by in situ hybridization on ultrathin frozen sections of pituitaries removed from rats injected with saline, GH-releasing factor (GRF) or somatostatin. A 4-fold increase in labeling of the anterior lobe was observed after GRF, no changes with somatostatin. After ultrastructural in situ hybridization, labeling with the GH cDNA probe was specific to somatotrophs. Two populations of cells containing few or many secretory granules were labeled mainly in the cytoplasm or in both cytoplasm and nucleus. Some cells showed labeling at the perinuclear membrane. Hybridization with the GH intron I probe showed the same cell specificity with silver grains mainly located in the nucleus. After GRF, sequences hybridizing to growth hormone cDNA were increased mainly in the nucleus of somatotrophs when compared to mock-injected rats, indirectly suggesting an increase in the transcriptional activity of the growth hormone gene. After somatostatin, the density of labeling in the nucleus was increased suggesting that somatostatin may prevent the export of growth hormone mRNA molecules from the nucleus to the cytoplasm.

Localization of mRNA coding for the three subtypes of atrial natriuretic factor (ANF) receptors in rat anterior pituitary gland cells.

Atrial Natriuretic Factor (ANF) action is mediated by highly selective and specific receptors. Three subtypes have been characterized and cloned: ANF receptor-A, -B and -C. These subtypes are all expressed in the anterior pituitary of the rat. In the present study, the mRNA for each subtype was detected by in situ hybridization. The amounts of ANFR-A and -B mRNA were found to be similar, and to be twice that of ANFR-C mRNA. At the ultrastructural level, the three types of ANFR mRNA were expressed in three anterior pituitary cell types, namely lactotrophs, corticotrophs, and gonadotrophs, identified by their hormonal content. No signal was revealed in somatotrophs or thyrotrophs. The different forms of mRNA were similar in terms of subcellular localization: in the cytoplasmic matrix and the nuclear euchromatin. These data indicate that the anterior pituitary is an important target tissue for ANF action.

In situ hybridization to rat brain and pituitary gland of growth hormone cDNA.

Using in situ hybridization, we have investigated the presence of mRNA coding for growth hormone (GH) in the rat brain. Using 32P-labeled GH cDNA as a probe, the pituitary gland showed hybridization in unfixed sections. Using 3H-labeled GH cDNA hybridized to fixed sections, only cells in the anterior pituitary were labeled in good agreement with the localization of somatotropes. In the brain, wide zones were labeled with 32P-GH cDNA: the caudate putamen, the striatum, the ventral thalamus, the formatio reticularis and the basal cortex. With the 3H GH-cDNA probe, more discrete regions of the brain showed hybridization: the basal cortex, the outside part of the hippocampus and the caudate putamen.

Chromosomal mapping of HCaRG, a novel hypertension-related, calcium-regulated gene.

We recently identified a novel gene that is negatively regulated by extracellular calcium concentration with higher levels of transcripts in hypertensive animals (SHR). We named this gene HCaRG (Hypertension-related, Calcium-Regulated Gene). In this work we report the chromosomal localization of the HCaRG gene among different species. We identified a BglII RFLP between BN.lx and SHR rats. We then analysed the strain distribution pattern of this RFLP in 31 RIS, originating from BN.lx and SHR rats, and compared it to the segregation of 475 markers localized in the rat genetic map. Hcarg localizes to the rat chromosome 7 between the markers Mit3 and Mit4. This region is homologous to human chromosome 8q21-24. We identified three clones in Genbank that contain the sequence of HCaRG. It was therefore possible to narrow down the localization of human HCaRG to chromosome 8q24.3. Furthermore, a suggestive localization of mouse Hcarg based on conservation of linkage between human and mouse is on chromosome 15. We previously identified a putative calcium-binding motif (EF-Hand) and a nuclear receptor-binding domain (LxxLL) in the rat sequence of the HCaRG protein. Sequence comparison between five different species showed that these domains are highly conserved. Furthermore, a search of ESTs in Genbank for homologous sequences showed that HCaRG is expressed only in eukaryotes, particularly in mammals.

Quantitative founder-effect analysis of French Canadian families identifies specific loci contributing to metabolic phenotypes of hypertension.

The Saguenay-Lac St-Jean population of Quebec is relatively isolated and has genealogical records dating to the 17th-century French founders. In 120 extended families with at least one sib pair affected with early-onset hypertension and/or dyslipidemia, we analyzed the genetic determinants of hypertension and related cardiovascular and metabolic conditions. Variance-components linkage analysis revealed 46 loci after 100,000 permutations. The most prominent clusters of overlapping quantitative-trait loci were on chromosomes 1 and 3, a finding supported by principal-components and bivariate analyses. These genetic determinants were further tested by classifying families by use of LOD score density analysis for each measured phenotype at every 5 cM. Our study showed the founder effect over several generations and classes of living individuals. This quantitative genealogical approach supports the notion of the ancestral causality of traits uniquely present and inherited in distinct family classes. With the founder effect, traits determined within population subsets are measurably and quantitatively transmitted through generational lineage, with a precise component contributing to phenotypic variance. These methods should accelerate the uncovering of causal haplotypes in complex diseases such as hypertension and metabolic syndrome.

Regulation of growth hormone mRNA and pro-opiomelanocortin mRNA levels by cyclic AMP in rat anterior pituitary cells in culture.

To understand the hormonal regulation of steady-state growth hormone (GH) mRNA levels, we investigated the interaction between somatostatin and agents known to increase intracellular cAMP activity on GH mRNA, GH synthesis, cell content of GH, and GH release in primary cultures of rat anterior pituitary cells. We simultaneously studied the modulation of the steady-state of pro-opiomelanocortin (POMC) mRNA levels by cAMP. In four independent experiments, a 48-hr exposure to 0.3 mM 3-isobutyl-1-methylxanthine (IBMX) or 3 mM 8-bromoadenosine 3',5'-cyclic monophosphate (8Br-cAMP) increased GH mRNA levels from 70 to 170% and from 70 to 150% above control (p less than 0.001), respectively. Parallel increases in GH release accompanied by a corresponding decrease in the intracellular content of GH were also obtained. Following a 48-hr incubation with 100 nM somatostatin alone, no change in GH mRNA levels was observed whereas GH release was inhibited by 90%, GH cell content doubled, and GH synthesis decreased by 40%. Surprisingly, somatostatin potentiated the enhancing effect of 8Br-cAMP on GH mRNA levels. In the same cell preparation, a 48-hr exposure to IBMX or 8Br-cAMP stimulated adrenocorticotropin release by 9.4- and 18.0-fold, respectively, and increased POMC mRNA levels by 2.4- and 2.3-fold (p less than 0.001), respectively. No change in beta-actin mRNA was observed after these treatments. These data indicate that increased intracellular cAMP concentrations increase the steady-state level of GH mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)