Inhibition of atrial natriuretic peptide secretion by forskolin in noncontracting cultured atrial myocytes

Secretory rates for immunoreactive atrial natriuretic peptide (ANP) by 7 - 8 day-old primary cultures of atrial myocytes from adult rats (with myocyte contraction inhibited by tetrodotoxin (TTX)) were (a) constant for at least two hours, and (b) significantly slowed by forskolin (1, 5, and 25 microM), dibutyryl cyclic adenosine monophosphate (1 mM), or isobutylmethylxanthine (100 microM). The substantial rates of ANP secretion which persisted in cells rendered noncontracting either by inhibiting Ca2+ influx via reduction of external [Ca2+] to less than 10(-7) M or by inhibiting sarcoplasmic reticulum Ca2+ release with 100 microM ryanodine were significantly slowed by 25 microM forskolin, but forskolin sensitivity was lost by cells exposed simultaneously to external Ca2+ concentration of less than 10(-7) M and 100 microM ryanodine. Quiescent myocytes whose ANP secretory rate was depressed by forskolin remained responsive to secretory stimulation by phorbol ester.     

The rat thymus--a site of atrial natriuretic peptide synthesis

The atrium of the heart has been demonstrated to represent the major site of synthesis of atrial natriuretic peptide (ANP), a potent natriuretic, diuretic and vasoactive hormone. Our recent studies revealed ANP-like material outside the heart, namely, in lymphoid follicles of the intestine and in the thymus, and now we report data demonstrating the thymus as a site of synthesis for ANP. The experimental evidence is as follows: firstly, the immunoreactive material detected corresponds chromatographically with the precursor of ANP. Secondly, the thymus contains mRNA for ANP. Thirdly, immunohistochemistry locates ANP-like material to cortical thymocytes with particularly dense staining in the subcapsular areas of the thymus. Interestingly, both ANP-like material and the mRNA coding for ANP were expressed to a larger extent in newborn rats as compared to adult animals, suggesting that ANP may be involved in the development and/or function of T-cells.     

Stimulation of cyclic GMP synthesis in human cultured glomerular cells by atrial natriuretic peptide

Recently a stimulatory effect of atrial natriuretic peptide (ANP) on the particulate guanylate cyclase system has been reported in the glomeruli from different species. Using cultures of homogeneous human glomerular cell lines, we found that rat and human ANP stimulated markedly cGMP formation in epithelial cells with a threshold dose of 1 nM. A 20-fold increase was obtained at 5 microM. Stimulation was also present but less substantial (2-fold at 5 microM) in mesangial cells. cGMP was formed rapidly and released in the medium. ANP and sodium nitroprusside, an activator of soluble guanylate cyclase, had additive effects on cGMP formation. ANP did not inhibit cAMP formation in both cell lines. These results demonstrate that, at least in the human species, epithelial cells represent the main target of ANP in the glomerulus. Synthesis of cGMP in the glomerular epithelial cells in response to ANP also suggests that the excess of urinary cGMP produced by the kidney which is observed after ANP administration is of glomerular rather than of tubular origin.     

Brain natriuretic peptide interacts with atrial natriuretic peptide receptor in cultured rat vascular smooth muscle cells

The effect of synthetic porcine brain natriuretic peptide (pBNP), a novel brain peptide with sequence homology to alpha-human atrial natriuretic peptide (hANP), on receptor binding and cGMP generation, was studied in cultured rat vascular smooth muscle cells (VSMC) and compared with that of alpha-hANP. 125I-pBNP bound to the cells in a time-dependent manner similar to that of 125I-alpha-hANP. Scatchard analysis indicated a single class of binding sites for pBNP with affinity and capacity identical to those of alpha-hANP. pBNP and alpha-hANP were almost equipotent in inhibiting the binding of either radioligand and stimulating intracellular cGMP generation. These data indicate that BNP and ANP interact with the same receptor sites to activate guanylate cyclase in rat VSMC.     

Functional multiplicity of atrial natriuretic peptide receptors on cultured rat Leydig tumor cells

Native rat atrial natriuretic peptide (NANP) was shown to bind with high affinity and to increase intracellular levels of cGMP in cultured rat Leydig tumor cells. A linear analog of NANP which lacks the disulfide-linked bridge structure also bound with high affinity but did not increase levels of intracellular cGMP or antagonize the increase of this cyclic nucleotide by NANP. These data are consistent with the existence of two functional subpopulations of ANP receptors on cultured rat Leydig tumor cells; one which is capable of activating guanylate cyclase and one which is not linked to this enzyme.     

Differential synthesis by cultured atrial and ventricular rat cardiac myocytes of myosin light chain isoforms

Dissociated cells from neonatal rat atria and ventricles were cultured in monolayers for 3 days. Newly synthesized 35S-methionine labeled myosin light chain isoforms ALC-1, ALC-2 (atrial) and VLC-1, VLC-2 (ventricular) were identified on 2D gels, and their pattern of synthesis was compared to that of myocard fragments immediately after explanation. ALCs were synthesized in 5- to 10-fold excess over VLCs by atrial cultures, whereas the converse was true for ventricular cultures, with two exceptions: one third of the LC-1 synthesized by ventricular fragments was ALC-1, and dissociated atrial cells synthesized very little LC-2 of either isoform. The former finding corresponds to the relatively high proportion of ALC-1 in neonatal ventricular tissue. We conclude that the regional programme of LC isoform expression is basically retained after tissue explantation and even after dissociation and culturing of cardiac myocytes.     

Immunoreactive atrial natriuretic peptide in the thyroid gland

Human thyroid follicles and primary cell cultures derived from them demonstrated atrial natriuretic peptide (ANP)-like immunoreactivity when stained with a monoclonal antibody raised against rat alpha-ANP (ANP 1-28). In thyroid sections the staining was most intense in the tall cuboidal epithelium of small follicles. The intracellular distribution of immunoreactive (ir)-ANP in primary cultures of thyroid follicular cells consisted of discrete granules with a largely perinuclear distribution. The granule density increased with time in culture but was unaffected by exogenous ANP, suggesting an intrinsic synthesis of the immunoreactivity. Thyroid stimulating hormone (TSH; thyrotropin) failed to alter the distribution of ir-ANP after either short-term (6 h) or long-term (1-12 day) exposure. Epinephrine or norepinephrine treatment, however, caused a reduction in the ir-ANP granularity compared with controls in what might represent a stimulated release of the immunoreactivity. The present results suggest that the peptide ANP coexists with thyroid hormones in follicular cells and that the two endocrine activities might be under separate control mechanisms.     

Stimulation of atrial natriuretic peptide secretion and synthesis by Na-K-ATPase inhibitors

Ouabain has been reported to increase the secretion of ANP in vitro. In this study, we focused on whether this action is common in Na-K-ATPase inhibitors (ATPI) and whether ATPI simply increase the release of ANP or stimulate both its biosynthesis and release. The effects of ouabain and digoxin on secretion of ANP and accumulation of ANP mRNA were investigated in the rat cardiocyte superfusion system. Ouabain and digoxin increased the immunoreactive ANP (iANP) output into perfusate and accumulation of ANP mRNA significantly. These results suggest that ATPI may stimulate both ANP biosynthesis and release in vitro.     

Arachidonic acid metabolites regulate the secretion of atrial natriuretic peptide in cultured rat atrial cardiocytes.

Prostaglandins F2 alpha and E2 increase release of immunoreactive (irANP) in primary cultures of rat atrial cardiocytes. This effect is independent of cell density in the cultures and does not appear to operate through a cAMP-dependent mechanism. Studies that probed the PGF2 alpha effect with a number of different pharmacological antagonists suggest that it is tied to a calmodulin-dependent step. This latter effect does not appear to be related to increased calcium entry through voltage-gated channels in the plasma membrane nor to mobilization of ryanodine-sensitive intracellular calcium pools. Inhibitors of the lipoxygenase pathway, a second avenue of arachidonate metabolism, resulted in a decrease in irANP release from cultured atrial or ventricular cardiocytes. Leukotriene C4, a lipoxygenase product, had a modest effect to promote irANP release over a 24-h period. However, pretreatment of anesthetized rats with nordihydroguarietic acid, a lipoxygenase inhibitor, had no effect on stretch-dependent release of irANP from the heart in vivo. These findings suggest that the prostaglandins represent the more important group of arachidonate metabolites in regulating irANP release physiologically.     

Modulation by NaCl of atrial natriuretic peptide receptor levels and cyclic GMP responsiveness to atrial natriuretic peptide of cultured vascular endothelial cells.

Type C atrial natriuretic peptide (ANP) receptor levels in cultured vascular endothelial cells were found to be very sensitive to NaCl and shown to be inversely related to the magnitude of ANP-induced cGMP response of the cells. Endothelial cells from bovine carotid artery were subcultured in Eagle's minimum essential medium supplemented with 10% fetal bovine serum (MEM-FBS) and in MEM-FBS plus 25 and 50 mM NaCl. Determination, after several passages, of ANP receptor levels in these cells by 125I-ANP binding assay and affinity labeling revealed a marked reduction in the number of type C receptor in the NaCl-treated cells, whereas type A receptor density was not affected. RNase protection assay to estimate the levels of type C receptor mRNA indicated that the reduction occurred at a pre-translational level. In spite of the decrease in type C receptor number and no significant change in type A receptor (i.e. particulate guanylate cyclase) levels, cGMP response of the NaCl-treated cells to ANP was greatly exaggerated; this sensitization was also observed in membrane preparations. Simple masking of type C ANP receptor with C-ANF (des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANP), a ring-deleted ANP analog, did not produce any sensitization of the cGMP response to ANP; therefore, the above phenomenon cannot simply be explained by the clearance function of the type C receptor. Although whether the type C receptor depletion is directly related to the sensitization of the type A receptor/cyclase is not known, the phenomenon reported and characterized here will serve as a useful basis for elucidating ANP receptor regulation and activation.     

Binding of synthetic beta-human atrial natriuretic peptide to cultured rat vascular smooth muscle cells

We have studied the effects of synthetic beta-human atrial natriuretic peptide (beta-hANP), an antiparallel dimer of alpha-hANP, on receptor binding and cGMP generation in cultured rat vascular smooth muscle cells and compared the effects with those of alpha-hANP. Characteristics of temperature-dependent binding and degradation of 125I-beta-hANP were similar to those of 125I-alpha-hANP. Scatchard analysis indicated a single class of binding sites for beta-hANP with a maximal binding capacity one-half that of alpha-hANP. Parallel and antiparallel dimers were equipotent in inhibiting the binding and stimulating intracellular cGMP formation, of which the maximal effect was about one-half that of alpha-hANP. Reverse-phase high performance liquid chromatography revealed that most of beta-hANP added to cells was converted to a small molecular mass component corresponding to alpha-hANP after incubation. These data suggest that the less potent effect of beta-hANP in receptor binding and cGMP generation may be partly accounted for by the possible conversion of beta-hANP to alpha-hANP at the site of target cells.     

Plasma atrial natriuretic peptide in states of altered thyroid function

To examine a possible role of atrial natriuretic peptide (ANP) in water and electrolyte disturbances associated with thyroid disorders, plasma ANP levels were studied in patients with hyper- and hypothyroidism. In 5 of the 21 hyperthyroid patients, including two patients with atrial fibrillation and two patients with mild cardiomegaly, the plasma ANP concentration was increased when compared to normal subjects. After treatment with methimazole or propylthiouracil, the plasma ANP concentration fell to normal in 4 patients, while it remained high in one patient who had persistent atrial fibrillation. No significant correlation was found between plasma ANP and the heart rate in untreated hyperthyroid patients. Plasma ANP was within the normal range in all 8 patients with hypothyroidism. During treatment with T4, the plasma ANP concentration increased in 6 of the 7 patients. Chest X-ray films and ultrasonic echocardiography demonstrated pericardial effusion in 4 of these patients before therapy. A weak but significant correlation was found between the plasma ANP and T4 concentration, and between plasma ANP and free T4 in hyper- and hypothyroid patients before and after treatment. These results indicate that abnormalities in ANP dynamics in thyroid disorders may probably be caused by hemodynamic changes resulting from a thyroid hormone excess or deficiency.     

Distribution of atrial natriuretic peptide and its effects on contraction and intracellular calcium in ventricular myocytes from streptozotocin-induced diabetic rat

The distribution of atrial natriuretic peptide (ANP) in blood plasma and cardiac muscle and its effects on ventricular myocyte contraction and intracellular free calcium concentration [Ca2+]i in the streptozotocin (STZ)-induced diabetic rat have been investigated. Blood plasma concentration and heart atrial and ventricular contents of ANP were significantly increased in STZ-treated rats compared to age-matched controls. STZ treatment increased the number of ventricular myocytes immunolabeled with antibodies against ANP. In control myocytes the percentage of cells that labeled positively and negatively were 17% versus 83%, respectively. However, in myocytes from STZ-treated rat the percentages were 52% versus 53%. Time to peak (TPK) shortening was significantly and characteristically prolonged in myocytes from STZ-treated rats (360+/-5 ms) compared to controls (305+/-5 ms). Amplitude of the Ca2+ transient was significantly increased in myocytes from STZ-treated rats compared to controls (0.39+/-0.02 versus 0.29+/-0.02 fura-2 RU in controls) and treatment with ANP reduced the amplitude of the Ca2+ transient to control levels. ANP may have a protective role in STZ-induced diabetic rat heart.     

Stimulation of Na-K-Cl cotransport in cultured vascular endothelial cells by atrial natriuretic peptide

Vascular endothelial cells have been shown to contain atrial natriuretic peptide (ANP)-sensitive Na-K-Cl cotransport system whose activity is regulated by intracellular cGMP levels. Addition of ANP to culture medium stimulated 86Rb+ uptake in bovine endothelial cells with a concomitant increase in cGMP contents. This action of ANP was mimicked by 8-bromo-cGMP and completely diminished by furosemide. These results indicate that ANP selectively activates the Na-K-Cl cotransporter in vascular endothelial cells via cGMP and offer new insight into the physiological significance of endothelial ANP receptors.     

Effects of atrial natriuretic peptide in the gut

The intestinal tract is a target organ for atrial natriuretic peptide (ANP), characterized by various biologic activities, immunoreactivity, as well as specific binding sites for ANP. A review of previous studies reveals that ANP is an important regulator of water and nutrient intake, which acts via multiple signaling pathways including activation of guanylyl cyclase to produce its biologic responses. As a regulator, the peptide locally controls hydrosaline balance and acute systemic effects. Therefore, ANP could also act as a local mediator or paracrine effector of intestinal function.     

Microvascular effects of atrial natriuretic peptide in rat cremaster

Experiments utilized the open cremaster preparation to test the hypothesis that atrial natriuretic peptide (ANP)-induced volume changes result from microvascular resistance alterations. Atrial natriuretic peptide (25, 100, and 500 ng/kg/min, IV) or vehicle was infused into anesthetized rats. At the two highest ANP infusion rates, mean arterial pressure was significantly reduced from 104 ± 3 (control) to 87 ± 2 and 77 ± 2 mmHg, respectively. Hematocrit was 41.0 ± 0.8 and 45.6 ± 0.9% (p < 0.05) at the end of vehicle and ANP infusions, respectively. Despite these effects of ANP, there were no significant arteriolar or venular diameter alterations. Thirty μM nitroprusside significantly dilated all vessel segments except large venules. These observations suggest that resistance alterations in the skeletal muscle microvasculature are not the cause of ANP-induced fluid movement.     

Secretion of atrial natriuretic peptide (ANP) from fish atrial and ventricular myocytes in tissue culture

Primary cultures of atrial and ventricular myocytes (approx. 1 x 10(5) cells/culture) were prepared from adult teleost fish Gila atraria and maintained for 10 days. Immunoreactive atrial natriuretic peptide (ir-ANP) from fish atrial and ventricular cells was 3.9 and 2.8 ng/culture respectively, values not significantly different. Atriocytes from rat and mouse secreted comparable amounts of ANP which were not significantly different from atrial fish cultures (5.2 and 4.3 ng/culture). In contrast, their ventricular myocytes secreted only small quantities of ANP (0.8 and 0.3 ng/culture). When analyzed by reversed-phase HPLC, the media of both fish atrial and ventricular myocytes contained a peptide which exhibited properties similar to authentic human ANP (Ser 99-Tyr 126), suggesting a significant degree of sequence homology between fish and mammalian ANP. Fish ventricular cells, unlike normal mammalian ventricular cells, secrete substantial quantities of immunoreactive-ANP.     

Regulation of SBP synthesis in human cancer cell lines by steroid and thyroid hormones

The presence of human sex steroid binding protein (SBP) in liver cells, the supposed site of SBP synthesis, and in other target cells for sex steroid hormones such as breast, endometrium and prostate epithelium, have been demonstrated by indirect immunofluorescence. It is not known whether SBP enters endometrial and prostate cells by endocytosis, possibly mediated by a cell membrane receptor process, or if SBP is synthesized in these cells. SBP mRNA has been searched in human cancer cell lines originated from liver (Hep G2/H5A), breast (MCF-7), endometrium (RL95-2) and prostate (LNCaP). It was only found in hepatoma cells where it is regulated by estradiol, antiestrogen tamoxifen and triiodothyronine, in a similar way as secreted SBP. This work provides evidence that human SBP is synthesized in the liver, and it also suggests that its regulation may involve non-steroidal factors.     

The cosecretional maturation of atrial natriuretic factor by primary atrial myocytes.

Cardiac myocytes store the 126-amino acid precursor of atrial natriuretic factor (pro-ANF), yet the mature, bioactive 28-amino acid peptide, ANF-(99-126), and the resulting N-terminal product, ANF-(1-98), are the forms of the hormone that are released by the heart and found in the circulation. Although previous studies have shown that the maturation of ANF takes place in the heart, it is not known whether it occurs in or on the myocyte concurrently with secretion, or whether cleavage takes place postsecretionally on either the myocyte surface or the surface of a nonmuscle cardiac cell. To address these questions, experiments were carried out in the present study using primary atrial cultures that had been prepared such that greater than 90% of the cells were myocytes. Reversed-phase and ion-exchange HPLC, coupled with immunoprecipitation of biosynthetically labeled ANF, showed that the stored peptide, pro-ANF, was cleaved between residues 98 and 99 such that ANF-(1-98) and (99-126) accumulated in the medium. Coupling biosynthetic labeling with timed secretion experiments showed that the extent of ANF processing was not dependent on the time after secretion; maximal levels of processing were observed at all secretion times examined. Additionally, the processing-competent myocyte-enriched cultures were unable to cleave exogenously added pro-ANF. These results indicate that the myocyte is the cell type responsible for pro-ANF maturation and that this cleavage event takes place cosecretionally.