Nitric oxide increases fluid extravasation from the splenic circulation of the rat

We hypothesized that nitric oxide (NO) contributes to intrasplenic fluid extravasation by inducing greater relaxation in splenic resistance arteries than veins such that intrasplenic microvascular pressure (P(C)) rises. Fluid efflux was estimated by measuring the difference between splenic blood inflow and outflow. Intrasplenic infusion of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) (0.3 microg. 10 microl(-1). min(-1)) caused a significant increase in intrasplenic fluid efflux (baseline: 0.8 +/- 0.4 ml/min, n = 10 vs. peak rise during SNAP infusion: 1.3 +/- 0.4 ml/min, n = 10; P < 0.05). Intrasplenic P(C) was measured in the isolated, blood-perfused rat spleen. Intrasplenic infusion of SNAP (0.1 microg. 10 microl(-1). min(-1)) caused a significant increase in P(C) (saline: 10.9 +/- 0.2 mmHg, n = 3 vs. SNAP: 12.2 +/- 0.2 mmHg, n = 3; P < 0.05). Vasoreactivity of preconstricted splenic resistance vessels to sodium nitroprusside (SNP) (1 x 10(-12)-1 x 10(-4) M) and SNAP (1 x 10(-10)-3 x 10(-4) M) was investigated with the use of a wire myograph system. Significantly greater relaxation of arterioles than of venules occurred with both SNP (%maximal vasorelaxation: artery 96 +/- 2.3, n = 9 vs. vein 26 +/- 1.9, n = 10) and SNAP (%maximal vasorelaxation: artery 50 +/- 3.5, n = 11 vs. vein 32 +/- 1.7, n = 8). These results are consistent with our proposal that differential vasoreactivity of splenic resistance arteries and veins to NO elevates intrasplenic P(C) and increases fluid extravasation into the systemic lymphatic system.     

The effect of angiotensin II and ADH on the secretion of atrial natriuretic factor

Studies in intact animals have suggested that angiotensin II (AII) and antidiuretic hormone (ADH) increase the plasma concentration of atrial natriuretic factor (ANF). The purpose of these studies was to examine the effects of AII and ADH on ANF secretion in a rat heart-lung preparation under conditions where aortic pressure could be regulated and other indirect effects of these hormones eliminated. ANF secretion was estimated as the total amount of ANF present in a perfusion reservoir at the end of each 30-min period. A pump was used to deliver a fluorocarbon perfusate to the right atrium at rates of either 2 or 5 ml/min. In a time control series where venous return was maintained at 2 ml/min for three 30-min periods ANF secretion was 672 +/- 114, 794 +/- 91, and 793 +/- 125 pg/min (n = 6, P greater than 0.05). When venous return was increased from 2 to 5 ml/min ANF secretion increased from 669 +/- 81 to 1089 +/- 127 pg/min (P less than 0.01). The addition of AII to the perfusate in concentrations of 50, 100, or 200 pg/ml (n = 6 in each group) had no significant effect on basal ANF secretion or the ANF response to increasing venous return. Similarly, the addition of ADH to the perfusate in concentrations of 5, 25, or 100 pg/ml had no significant effect on ANF release from the heart. These results suggest that the ability of AII and ADH to increase plasma ANF concentration in vivo may be due to the effects of these hormones on right or left atrial pressure.     

Effect of nitric oxide synthase inhibition on cardiovascular and hormonal regulation during pregnancy in the rat

Recent studies have shown that nitric oxide (NO) biosynthesis increases in pregnancy and that inhibition of nitric oxide synthase (NOS) induces some pathological processes characteristic of preeclampsia. The current project sought to study the effect of the NOS inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME, 10 microg x min(-1), sc for 7 days) on plasma volume, plasma atrial natriuretic factor (ANF), plasma endothelin-1 (ET), and plasma renin activity (PRA) during gestation in conscious rats. NOS inhibition caused mean arterial pressure to increase in both virgin and 21-day pregnant rats. Plasma volume fell in the pregnant rats [L-NAME, 4.5 +/- 0.3 mL x 100 g(-1) body wt. (n = 7) vs. D-NAME, 6.8 +/- 0.2 mL x 100 g(-1) body wt. (n = 10); P < 0.05] but not in the virgin rats [L-NAME, 4.3 +/- 0.1 mL x 100 g(-1) body wt. (n = 6) vs. D-NAME, 4.8 +/- 0.2 mL x 100 g(-1) body wt. (n = 8)]. There was no effect of NOS inhibition on plasma ANF levels or PRA in either the virgin or pregnant rats. However, L-NAME did decrease plasma ET levels in the pregnant rats [L-NAME, 19.6 +/- 1.6 pg x mL(-1) (n = 8) vs. D-NAME, 11.6 +/- 2.5 pg x mL(-1) (n = 9); P < 0.05]. Our results confirm that NO is involved in cardiovascular homeostasis in pregnancy; NOS inhibition selectively reduces plasma volume in pregnant rats, thus mimicking a major pathophysiological perturbation of preeclampsia. However, it does not induce the hormonal changes characteristic of preeclampsia, namely the decrease in PRA and increase in plasma ET and ANF levels.     

Atrial natriuretic factor inhibits the CRH-stimulated secretion of ACTH and cortisol in man.

Corticotrophic secretion of ACTH is stimulated by corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP), and suppressed by glucocorticoids. In vitro and preclinical studies suggest that atrial natriuretic factor (ANF) may be a peptidergic inhibitor of pituitary-adrenocortical activity. The aim of this study was to elucidate a possible role of ANF as a modulator of ACTH release in humans. A bolus injection of 100 micrograms human CRH (hCRH) during a 30 min intravenous infusion of 5 micrograms/min human alpha atrial natriuretic factor (h alpha ANF) was administered at 19:00 to six healthy male volunteers. In comparison to saline, a blunted CRH-stimulated secretion of ACTH (mean maximum plasma level +/- SD 45 min after hCRH: saline 46.2 +/- 14.2 pg/ml, h alpha ANF 34.6 +/- 13.8 pg/ml, p-value = 0.007) and a delayed rise (10 min) in cortisol were detected. The maximum plasma cortisol levels remained nearly unchanged between saline and h alpha ANF administration (mean maximum plasma level +/- SD 60 min after hCRH: saline 182 +/- 26 ng/ml, h alpha ANF 166 +/- 54 ng/ml). No effects of h alpha ANF on basal cortisol levels were observed; in contrast, basal ACTH plasma levels were slightly reduced. Basal blood pressure and heart rate remained unaffected. In the control experiment, infusion of 3 IU AVP in the same experimental paradigm increased basal and stimulated ACTH and cortisol levels significantly in comparison to saline. These observations suggest that intravenously administered haANF inhibits the CRH-stimulated release of ACTH in man.     

Cellular signals regulating the release of ANF.

Atrial natriuretic factor (ANF), a peptide hormone that regulates salt and water balance and blood pressure, is synthesized, stored, and secreted from mammalian myocytes. Stretching of atrial myocytes stimulates ANF secretion, but the cellular processes involved in linking mechanical distension to ANF release are unknown. We reported that phorbol esters, which mimic the action of diacylglycerol by acting directly on protein kinase C and the Ca2+ ionophore A23187, which introduces free Ca2+ into the cell, both increase basal ANF secretion in the isolated perfused rat heart. Phorbol ester also increased responsiveness to Ca2+ channel agonists, such as Bay k8644, and to agents that increase cAMP, such as forskolin and membrane-permeable cAMP analogs. In neonatal cultured rat atrial myocytes, protein kinase C activation by 12-O-tetradecanoylphorbol 13-acetate stimulated ANF secretion, whereas the release was unresponsive to changes in intracellular Ca2+. Endothelin, which stimulates phospholipase C mediated hydrolysis of phosphoinositides and activates protein kinase C, increased both basal and atrial stretch-induced ANF secretion from isolated perfused rat hearts. Similarly, phorbol ester enhanced atrial stretch-stimulated ANF secretion, while the increase in intracellular Ca2+ appeared to be negatively coupled to the stretch-induced ANF release. Finally, phorbol ester stimulated ANF release from the severely hypertrophied ventricles of hypertensive animals but not from normal rat myocardium. These results suggest that the protein kinase C activity may play an important role in the regulation of basal ANF secretion both from atria and ventricular cells, and that stretch of atrial myocytes appears to be positively modulated by phorbol esters.     

Atrial natriuretic factor: radioimmunoassay and effects on adrenal and pituitary glands

A simple and sensitive radioimmunoassay was developed for measurement of immunoreactive atrial natriuretic factor (IR-ANF) in rat and human plasma and in rat atria. The two atria contain about 20 micrograms ANF per rat. The right atrium contained 2.5 times more ANF than did the left. Ether anesthesia and morphine markedly increased IR-ANF in rat plasma. The concentration of IR-ANF in plasma of clinically normal human subjects was 65.3 +/- 2.5 pg/ml. Paroxysmal tachycardia and rapid atrial pacing significantly increased IR-ANF in human plasma. Two- to seven-fold higher concentrations were found in coronary sinus blood than in the peripheral circulation. In the plasma of rats and humans, circulating ANF is probably a small-molecular-weight peptide. ANF acts on the adrenal and the pituitary. ANF inhibits aldosterone secretion from rat zona glomerulosa and steroid secretion by bovine adrenal zona glomerulosa and fasciculata. ANF stimulated the basal secretion of arginine vasopressin (AVP) in vitro and inhibited KCl-stimulated release of AVP.     

Cyclic AMP and cyclic GMP independent stimulation of ventricular calcium current by peroxynitrite donors in guinea pig myocytes

We investigated the potential involvement of peroxynitrite (ONOO(-)) in the modulation of calcium current (I(Ca)) in guinea pig ventricular myocytes with the whole-cell patch clamp technique and with cyclic AMP (cAMP) measurements. Because of the short half-life of ONOO(-) at physiological pH, we induced an increase in its intracellular levels by using donors of the precursors, nitric oxide (NO) and superoxide anion (O(2) (-)). High concentrations of NO donors, SpermineNONOate (sp/NO, 300 microM) or SNAP (300 microM) increased basal I(Ca) (50.3 +/- 4.6%, n = 7 and 46.2 +/- 5.0%, n = 13). The superoxide anion donor Pyrogallol (100 microM) also stimulated basal I(Ca) (44.6 +/- 2.8%, n = 11). At lower concentration sp/NO (10 nM) and Pyrogallol (1 microM), although separately ineffective on I(Ca), enhanced the current if applied together (33.5 +/- 0.7%, n = 7). The simultaneous donor of O(2) (-) and NO, SIN-1 (500 microM), also stimulated basal I(Ca) (22.8 +/- 2.1%, n = 13). In the presence of saturating cyclic GMP (cGMP, 50 microM) in the patch pipette or of extracellular dibutyryl cGMP (dbcGMP, 100 microM), I(Ca) was still increased by SIN-1 (32.0 +/- 6.1%, n = 4 and 30.0 +/- 5.4%, n = 8). Both Manganese(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP, 100 microM) a ONOO(-) scavenger, and superoxide dismutase (SOD) (150 U/ml) reversed the stimulatory effect of SIN-1 on I(Ca) (respectively -0.6 +/- 4.1%, n = 4 and 3.6 +/- 4.3%, n = 4). Intracellular cAMP level was unaltered by SIN-1, while it was enhanced by blocking the NO-cGMP pathway with the NO synthase inhibitor L-NMMA. These results suggest that peroxynitrite donors increase cardiac calcium current without the involvement of cAMP and cGMP.     

Alterations in atrial and plasma atrial natriuretic factor (ANF) content during development of hypoxia-induced pulmonary hypertension in the rat

Distension of the atrial wall has been proposed as a signal for the increased release of atrial natriuretic factor (ANF) from atrial myocytes in response to perceived volume overload. To determine whether pressure changes resulting from hypertension in the pulmonary circulation may stimulate release of ANF, rats were exposed to chronic hypobaric hypoxia for 3 or 21 days and the ANF concentration in the atria and plasma were determined by specific radioimmunoassay. Exposure to chronic hypoxia resulted in significant increases in hematocrit at both 3 (p less than 0.025) and 21 days (p less than 0.005) and in the development of right ventricular hypertrophy (RVH) expressed as the ratio of the weight of the right ventricle to the weight of the left ventricle and septum (RV/LV+S) at both 3 (RV/LV+S = 0.278 +/- 0.005) and 21 days (RV/LV+S = 0.536 +/- 0.021). After 21 days, left atrial (LA) ANF content was significantly increased in hypoxic rats compared to controls (508 +/- 70 ng/mg tissue vs 302 +/- 37 ng/mg), while right atrial (RA) ANF content was significantly reduced (440 +/- 45 vs 601 +/- 58 ng/mg). At this time, plasma ANF concentration was significantly elevated compared to controls (238 +/- 107 pg/ml vs 101 +/- 10 pg/ml). These results suggest that the development of pulmonary hypertension following chronic hypobaric exposure induces altered atrial ANF content and increased plasma ANF concentration as a result of altered distension of the atrial wall.     

Inhibition of nitric oxide synthase enhances superoxide activity in canine kidney

To evaluate the role of a potential interaction between superoxide anion (O(2)(-)) and nitric oxide (NO) in regulating kidney function, we examined the renal responses to intra-arterial infusion of a superoxide dismutase mimetic, tempol (0.5 mg.kg(-1).min(-1)), in anesthetized dogs treated with or without NO synthase inhibitor, N(omega)-nitro-l-arginine (NLA; 50 microg.kg(-1).min(-1)). In one group of dogs (n = 10), tempol infusion alone for 30 min before NLA infusion did not cause any significant changes in renal blood flow (RBF; 5.2 +/- 0.4 to 5.0 +/- 0.4 ml.min(-1).g(-1)), glomerular filtration rate (GFR; 0.79 +/- 0.04 to 0.77 +/- 0.04 ml.min(-1).g(-1)), urine flow (V; 13.6 +/- 2.1 to 13.9 +/- 2.5 microl.min(-1).g(-1)), or sodium excretion (U(Na)V; 2.4 +/- 0.3 to 2.2 +/- 0.3 micromol.min(-1).g(-1)). Interestingly, when tempol was infused in another group of dogs (n = 12) pretreated with NLA, it caused increases in V (4.4 +/- 0.4 to 9.7 +/- 1.4 microl.min(-1).g(-1)) and in U(Na)V (0.7 +/- 0.1 to 1.3 +/- 0.2 micromol.min(-1).g(-1)) without affecting RBF or GFR. Although NO inhibition caused usual qualitative responses in both groups of dogs, the antidiuretic (47 +/- 5 vs. 26 +/- 4%) and antinatriuretic (67 +/- 4 vs. 45 +/- 11%) responses to NLA were seen much less in dogs pretreated with tempol. NLA infusion alone increased urinary excretion of 8-isoprostane (13.9 +/- 2.7 to 22.8 +/- 3.6 pg.min(-1).g(-1); n = 7), which returned to the control levels (11.6 +/- 3.4 pg.min(-1).g(-1)) during coadministration of tempol. These data suggest that NO synthase inhibition causes enhancement of endogenous O(2)(-) levels and support the hypothesis that NO plays a protective role against the actions of O(2)(-) in the kidney.     

The influence of calcium on ANF release in the isolated rat atrium.

We developed an in vitro model of the isolated, perfused rat atrium with which to examine the mechanisms linking muscular stretch to atrial natriuretic factor (ANF) secretion. It was shown that an increase in atrial pressure causing distension of the atria is associated with a rise in ANF secretion correlating with the degree of pressure load. Pressure-induced ANF secretion is enhanced by the calcium blocker nifedipine or omission of calcium from the perfusion buffer. The changes in atrial volume in response to a given pressure load are also more pronounced in the absence of calcium or following the addition of the calcium blocker. These data suggest that in nonbeating atria, stretch-induced ANF secretion does not rely on calcium influx.     

Specific binding of atrial natriuretic factor to renal glomeruli during day or night antiorthostatic suspension in the rat

We observed a significant increase in plasma atrial natriuretic factor (ANF) in antiorthostatic hypokinetic suspension (AOH) rats after 2 h of suspension when the experiment was made during day. Plasma ANF was investigated in relation to renal glomerular ANF receptors during AOH at night. The aim of this study was 1) to compare the day and night ANF responses to AOH 2) to determine whether the renal glomerular ANF receptors are involved. The rats were divided into 2 groups: i) 24 population cage (PC), and ii) 24 were attached by the tail (Morey's model) and remained in the horizontal position (attached horizontal-AH). Six AH were suspended (30 degrees) for 2 hours (AOH) and sacrificed with the controls: PC and AH (12.00h). The same experiment was made during the night (24.00h). A significant increase in plasma ANF was found in both AOH and AH after 2 h of suspension during day and night (19 +/- 2.3 pg/ml vs 9 +/- 0.95 and 18 +/- 3 pg/ml vs 10.2 +/- 1.8 respectively). PC rats had a significantly higher ANF level (38 +/- 5 pg/ml) than AH or AOH. The glomerular ANF receptor population was slightly lower in AOH than in AH (429 +/- 12 fmol/mg protein vs 507 +/- 5) during day. During night, a significantly lower number of ANF receptors was observed in AOH animals as compared to AH (168 +/- 2 fmol/mg protein vs 455 +/- 3). A decrease in glomerular receptors was also noted in PC during night. Day-time head-down tilt, bed rest or head-out water induced a natriuretic and diuretic response, whereas the normal recumbency at night does not lead to such effects. We conclude that the natriuretic and diuretic response not observed during night was associated with elevated plasma ANF levels and decreased ANF receptor density.     

Role of endothelin receptor subtypes in volume-stimulated ANF secretion

The role of endothelin (ET) receptors was tested in volume-stimulated atrial natriuretic factor (ANF) secretion in conscious rats. Mean ANF responses to slow infusions (3 x 3.3 ml/8 min) were dose dependently reduced (P < 0.05) by bosentan (nonselective ET-receptor antagonist) from 64.1 +/- 18.1 (SE) pg/ml (control) to 52.6 +/- 16.1 (0.033 mg bosentan/rat), 16.1 +/- 7.6 (0. 33 mg/rat), and 11.6 +/- 6.5 pg/ml (3.3 mg/rat). The ET-A-receptor antagonist BQ-123 (1 mg/rat) had no effect relative to DMSO controls, whereas the putative ET-B antagonist IRL-1038 (0.1 mg/rat) abolished the response. In a second protocol, BQ-123 (>/=0.5 mg/rat) nonsignificantly reduced the peak ANF response (106.1 +/- 23.0 pg/ml) to 74.0 +/- 20.5 pg/ml for slow infusions (3.5 ml/8.5 min) but reduced the peak response (425.3 +/- 58.1 pg/ml) for fast infusions (6.6 ml/1 min) by 49.9% (P < 0.001) and for 340 pmoles ET-1 (328.8 +/- 69.5 pg/ml) by 83.5% (P < 0.0001). BQ-123 abolished the ET-1-induced increase in arterial pressure (21.8 +/- 5.2 mmHg at 1 min). Changes in central venous pressure were similar for DMSO and BQ-123 (slow: 0.91 and 1.14 mmHg; fast: 4.50 and 4.13 mmHg). The results suggest 1) ET-B receptors mainly mediate the ANF secretion to slow volume expansions of <1.6%/min; and 2) ET-A receptors mainly mediate the ANF response to acute volume overloads.     

Sulfonylurea receptor ligands modulate stretch-induced ANF secretion in rat atrial myocyte culture

Stretch-induced atrial natriuretic factor (ANF) secretion was studied in cultures of neonate atrial appendage myocytes. Stretch, applied for 40 min by hypotonic swelling, increased the mean area of 44 individually imaged myocytes by 4.8-8.8% (P < 0.0001) at 6 min and by 2.3-6.2% (P < 0.05) at 35 min. Stretch increased immunoreactive ANF release by 42% (P < 0.05) from a baseline of 315 pg/ml. The ATP-sensitive K(+) (K(ATP))-channel blocker tolbutamide (100 micromol/l) increased the stretch-stimulated release to 84% (P < 0.01) over baseline, whereas lower concentrations (1, 10, and 30 micromol/l) had no stimulatory effect. The K(ATP)-channel opener diazoxide (0.1, 1, 10, 30, and 100 micromol/l) inhibited stretch- plus tolbutamide-stimulated ANF release in a concentration-dependent manner, with IC(50) = 2.2 micromol/l, although 100 micromol/l diazoxide did not reduce the increase in mean cell area. The stretch-stimulated K(ATP) current, monitored in 82 whole cell recordings with sulfonylurea receptor (SUR) ligands, was inversely correlated with the stretch-induced ANF release (r(2) = 0.79, P < 0. 0001). In the absence of stretch, the K(ATP) current had no relationship with baseline ANF release, and baseline ANF release was not affected by the K(ATP)-channel modulators. The results show that SUR ligands that open K(ATP) channels inhibit stretch-induced ANF release in atrial myocytes, in correlation with the stretch-activated K(ATP) current. The subcellular site of action of the SUR ligands-plasmalemma or intracellular organelles-remains to be determined.     

Chronic nitric oxide synthase blockade desensitizes the heart to the negative metabolic effects of nitric oxide

The consequences of chronic nitric oxide synthase (NOS) blockade on the myocardial metabolic and guanylyl cyclase stimulatory effects of exogenous nitric oxide (NO) were determined. Thirty-three anesthetized open-chest rabbits were randomized into four groups: control, NO donor S-nitroso-N-acetyl-penicillamine (SNAP, 10(-4 )M), NOS blocking agent N(G)-nitro-L-arginine methyl ester (L-NAME, 20 mg/kg/day) for 10 days followed by a 24 hour washout and L-NAME for 10 days followed by a 24 hour washout plus SNAP. Myocardial O(2) consumption was determined from coronary flow (microspheres) and O(2) extraction (microspectrophotometry). Cyclic GMP and guanylyl cyclase activity were determined by radioimmunoassay. There were no baseline metabolic, functional or hemodynamic differences between control and L-NAME treated rabbits. SNAP in controls caused a reduction in O(2) consumption (SNAP 5.9+/-0.6 vs. control 8.4+/-0.8 ml O(2)/min/100 g) and a rise in cyclic GMP (SNAP 18.3+/-3.8 vs. control 10.4+/-0.9 pmol/g). After chronic L-NAME treatment, SNAP caused no significant changes in O(2) consumption (SNAP 7.1+/-0.8 vs. control 6.4+/-0.7) or cyclic GMP (SNAP 14.2+/-1.8 vs. control 12.1+/-1.3). In controls, guanylyl cyclase activity was significantly stimulated by SNAP (216.7+/-20.0 SNAP vs. 34.4+/-2.5 pmol/mg/min base), while this increase was blunted after L-NAME (115.9+/-24.5 SNAP vs. 24.9+/-4.7 base). These results demonstrated that chronic NOS blockade followed by washout blunts the response to exogenous NO, with little effect on cyclic GMP or myocardial O(2) consumption. This was related to reduced guanylyl cyclase activity after chronic L-NAME. These results suggest that, unlike many receptor systems, the NO-cyclic GMP signal transduction system becomes downregulated upon chronic inhibition.     

In vivo assessment of microvascular nitric oxide production and its relation with blood flow

To assess the hypothesis that microvascular nitric oxide (NO) is critical to maintain blood flow and solute exchange, we quantified NO production in the hamster cheek pouch in vivo, correlating it with vascular dynamics. Hamsters (100-120 g) were anesthetized and prepared for measurement of microvessel diameters by intravital microscopy, of plasma flow by isotopic sodium clearance, and of NO production by chemiluminescence. Analysis of endothelial NO synthase (eNOS) location by immunocytochemistry and subcellular fractionation revealed that eNOS was present in arterioles and venules and was 67 +/- 7% membrane bound. Basal NO release was 60.1 +/- 5.1 pM/min (n = 35), and plasma flow was 2.95 +/- 0.27 microl/min (n = 29). Local NO synthase inhibition with 30 microM N(omega)-nitro-L-arginine reduced NO production to 8.6 +/- 2.6 pmol/min (-83 +/- 5%, n = 9) and plasma flow to 1.95 +/- 0.15 microl/min (-28 +/- 12%, n = 17) within 30-45 min, in parallel with constriction of arterioles (9-14%) and venules (19-25%). The effects of N(omega)-nitro-L-arginine (10-30 microM) were proportional to basal microvascular conductance (r = 0.7, P < 0.05) and fully prevented by 1 mM L-arginine. We conclude that in this tissue, NO production contributes to 35-50% of resting microvascular conductance and plasma-tissue exchange.     

Basal and stimulated ANF secretion: role of tissue preparation

Our previous results showed that addition of agonists, such as vasopressin and angiotensin, added to incubation medium with freshly excised rat atria caused marked release of atrial natriuretic factor (ANF). This release was in the form of prohormone rather than active peptide. Since others had difficulty reproducing these findings, in the present study we investigated ANF release with and without angiotensin addition in two sets of atrial tissue. In the first, tissue was blotted and carefully cleaned as previously described; in the second, atrial tissue was placed into incubation medium without prior preparation. ANF activity in the medium was measured by radioimmunoassay and receptor assay. Using the immunoassay, basal release of ANF was threefold greater from prepared vs. nonprepared atrial tissue; significant stimulation by angiotensin was seen only in the prepared atria. ANF release measured by radioreceptor assay was 1/5-1/10 of that measured by immunoassay. Taking the difference between the two measurements as an index of prohormone secretion, the results confirm that both basal and stimulated release was primarily in the form of proANF. Scanning electron microscopy revealed that cleaning of the atria had removed the endocardial lining of the tissue. The results thus indicate that an intact endocardium can prevent agonist-induced proANF secretion, suggesting that this tissue may be an important modulator of plasma ANF levels.     

Behavior of atrial natriuretic factor in an experimental model of Trypanosoma cruzi infection in rats

Enhanced atrial natriuretic factor (ANF) production by the heart is related to hemodynamic overload, cardiac growth, and hypertrophy. The heart is one of the most affected organs during Trypanosoma cruzi infection. We tested the hypothesis that myocarditis produced by parasite infection alters the natriuretic peptide system by investigating the behavior of plasma ANF during the acute and chronic stages of T. cruzi infection in rats. Sprague-Dawley rats were infected with T. cruzi clone Sylvio-X10/7. Cardiac morphology showed damage to myocardial cells and lymphocyte infiltration in the acute phase; and fibrosis and cell atrophy in the chronic period. Plasma ANF levels (radioimmunoassay) were significantly higher in acute (348 +/- 40 vs. 195 +/- 36 pg/ml, P < 0.05, n = 17) and chronic T. cruzi myocarditis (545 +/- 81 vs. 229 +/- 38 pg/ml, P < 0.001, n = 11) than in their respective controls (n = 10 and 14). Rats in the chronic phase also showed higher levels than rats in the acute phase (P < 0.01). The damage of myocardial cells produced by the parasite and the subsequent inflammatory response could be responsible for the elevation of plasma ANF during the acute period of T. cruzi infection. The highest plasma ANF levels found in chronically infected rats could be derived from the progressive failure of cardiac function.     

Nitric oxide and platelet energy metabolism

This study was undertaken to determine whether nitric oxide (NO) can affect platelet responses through the inhibition of energy production. It was found that NO donors: S-nitroso-N-acetylpenicyllamine, SNAP, (5-50 microM) and sodium nitroprusside, SNP, (5-100 microM) inhibited collagen- and ADP-induced aggregation of porcine platelets. The corresponding IC50 values for SNAP and SNP varied from 5 to 30 microM and from 9 to 75 microM, respectively. Collagen- and thrombin-induced platelet secretion was inhibited by SNAP (IC50 = 50 microM) and by SNP (IC50 = 100 microM). SNAP (20-100 microM), SNP (10-200 microM) and collagen (20 microg/ml) stimulated glycolysis in intact platelets. The degree of glycolysis stimulation exerted by NO donors was similar to that produced by respiratory chain inhibitors (cyanide and antimycin A) or uncouplers (2,4-dinitrophenol). Neither the NO donors nor the respiratory chain blockers affected glycolysis in platelet homogenate. SNAP (20-100 microM) and SNP (50-200 microM) inhibited oxygen consumption by platelets. The effect of SNP and SNAP on glycolysis and respiration was not reduced by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, a selective inhibitor of NO-stimulated guanylate cyclase. SNAP (5-100 microM) and SNP (10-300 microM) inhibited the activity of platelet cytochrome oxidase and had no effect on NADH:ubiquinone oxidoreductase and succinate dehydrogenase. Blocking of the mitochondrial energy production by antimycin A slightly affected collagen-evoked aggregation and strongly inhibited platelet secretion. The results indicate that: 1) in porcine platelets NO is able to diminish mitochondrial energy production through the inhibition of cytochrome oxidase, 2) the inhibitory effect of NO on platelet secretion (but not aggregation) can be attributed to the reduction of mitochondrial energy production.     

High salt intake-induced changes in atrial natriuretic factor kinetics are mediated by clearance receptors.

We have reported a paradoxical plasma atrial natriuretic factor (ANF) decline following prolonged high salt intake that was attributed to an increased tissue uptake of circulating ANF, leading to its augmented distribution volume (Vas) and metabolic clearance rate (MCR) as compared with control rats on a standard diet. To explore this phenomenon further, we evaluated possible chronic salt-loading-induced changes in ANF clearance (C-ANF) receptors, which appear to play a major role in ANF removal from the circulation. We studied changes in plasma [125I]ANF(1-28) and its pharmacokinetics after preoccupation of C-ANF receptors by its specific ligand, C-ANF(4-23), in high-salt-treated rats and their controls. Following C-ANF(4-23) administration, we detected significantly higher circulating [125I]ANF levels throughout the study period (8 min) in high-salt-fed rats compared with the controls (280-470% vs 100-215% increase of basal values, P less than 0.05). C-ANF(4-23) infusion caused a significantly greater decrease of the metabolic clearance rate and distribution volume of [125I]ANF in high-salt-fed rats than in control animals (74 +/- 6% vs 41 +/- 6% and 75 +/- 4% vs 50 +/- 5% of basal values, respectively; P less than 0.05). These data suggest that a prolonged high salt diet may increase the availability of C-ANF receptors and, through this mechanism, may negatively modulate plasma ANF concentrations. C-ANF receptors may thus fulfill a regulatory function on circulating ANF during prolonged salt loading in rats.     

Effect of estradiol-17beta on PGF and total protein content in bovine uterine flushings and peripheral plasma concentration of 13, 14-dihydro-15-keto-PGF(2alpha)

Two experiments were conducted to examine the effect of estradiol-17beta (E(2)-17beta) on content of immunoreactive prostagladin F(2)alpha (PGF, ng) and total protein (TUP, mg) in uterine flushings, as well as concentrations of 13, 14-dihydro-15-keto-PGF(2)alpha (PGFM) in plasma (Pg/ml). In experiment 1, Holstein heifers were utilized in a single reversal trial in which either E(2)-17beta (3 mg in 2 ml saline/ethanol 50:50; n=5) or vehicle alone (n=6) were given intravenously on day 14 or 15 of the estrous cycle (Period 1) following an induced estrus (day of estrus = day 0). Treatment (Trt) groups were reversed in Period 2 (Day 14 or 15 of the second estrous cycle). Jugular venous plasma was obtained before treatment (Oh), and at 5, 6, and 9h posttreatment (PT). Uterine flushings were collected nonsurgically in vivo , per cervix, via Foley catheter at 6h PT (20 ml of .9% saline per uterine horn). E(2)-17beta did not significantly alter (E(2)-17beta vs vehicle; x(-) +/- S.E.M.) PGF (1674 +/- .11 +/- 338.39 vs 1889.91 +/- 400.24 ng; P> .10) or TUP (33.25 +/- 2.57 vs 39.16 +/- 3.04 mg; P > .10). However, E(2)-17beta increased (P < .05) plasma PGFM (E(2)-17beta vs vehicle) after treatment (0h, 113.2 vs 163.8; 5h, 312.5 vs 203.9; 6h, 324.5 vs 198.0; 9h, 323.2 vs 246.8, pg/ml). In experiment 2, crossbred beef cattle received comparable treatments of either E(2)-17beta (n=5) or vehicle (n=5) on day 14 or 15 postestrus. Jugular venous plasma was obtained at 0h PT, and at 6h PT. Uterine flushings (1.9% saline, 20 ml per uterine horn) and peripheral plasma were collected at slaughter. Estradiol-17beta increased PGF (30.07 +/- 5.94 vs 8.46 +/- 2.01 ng; P> <.05) in uterine flushings as well as PGFM in plasma (E(2)-17beta : 55.82 +/- 19.13 pg/ml, at 0h and 89.31 +/- 14.02 pg/ml, at 6h, vs saline: 103.46 +/- 50.73 pg/ml, at 0h and 17.78 +/- 14.22, at 6h). Estradiol-17beta stimulated uterine production and release of PGF and protein as measured in flushings (experiment 2) as well as plasma PGFM responses (experiments 1 and 2). Uterine and/or cervical stimulation of experiment 1 may have masked uterine response to E(2)-17beta.