Guillermo A. Zeballos

Pharmacodynamics of Plasma Nitrate/Nitrite as an Indication of Nitric Oxide Formation in Conscious Dogs

BACKGROUND The present investigation was undertaken to better understand the production of nitric oxide (NO) in vivo as measured by alterations in plasma nitrite or nitrate in blood samples from studies in experimental animals or clinical studies in humans. METHODS AND RESULTS Plasma samples were taken from the aorta, the coronary sinus, a peripheral vein in the leg (skeletal muscle), or the right ventricle (mixed venous) in chronically instrumented conscious dogs. Plasma nitrite was converted to NO gas in an argon environment by use of hydrochloric acid, and plasma nitrate was converted first to nitrite with nitrate reductase and then to NO gas with acid. Standard curves were constructed, and the amount of nitrite and nitrate in plasma was determined. The primary metabolite was nitrate, whereas nitrate was approximately 10% of the total and remained constant. In the resting dog, the only vascular bed with a positive arterial-venous nitrate difference, evidence for production of NO, was the heart. Nitrate infusion into quietly resting dogs resulted in increases in plasma nitrate up to 38 +/- 3.4 mmol/L, increases in systemic arterial pressure, and a marked diuresis. The plasma half-life was calculated as 3.8 hours. The volume of distribution was calculated as 0.215 L/kg, or equivalent to the extracellular volume. CONCLUSIONS These studies indicate that nitrate is a reliable measure of NO metabolism in vivo but that because of the long half-life, nitrate will accumulate in plasma once it is produced. Because of the large volume of distribution (21% of body weight versus the 4% of body weight usually attributed to plasma volume, the compartment in which nitrate is measured), simple measures of plasma nitrate underestimate by a factor of 4 to 6 the actual production of nitrate or NO by the body. In disease states, such as heart failure, in which renal function and extracellular volume are altered, caution should be exercised when increases in nitrate in plasma as an index of NO formation are evaluated.

Role of nitric oxide and endothelin-1 in monocrotaline-induced pulmonary hypertension in rats

OBJECTIVE Nitric oxide (NO) and endothelin-1 (ET-1) have both been implicated in the pathogenesis of pulmonary hypertension (PH). Therefore, we examined NO-related relaxation and ET-1 levels in rat hilar pulmonary arteries (PA) during the progression of monocrotaline (MCT)-induced PH. METHODS Rats were studied 1 and 2 weeks after a single subcutaneous injection of MCT (80 mg/kg). Pulmonary artery pressure (PAP), right ventricular hypertrophy (RVH), NO-related relaxation and tissue ET-1 levels in PA were evaluated and compared with control (C). RESULTS One week post-MCT, endothelium (E)-dependent relaxation to 10(-5) M adenosine diphosphate (ADP), 10(-5) M A23187 and 10(-5) M acetylcholine (ACh) and tissue ET-1 levels in PA were normal. Rats in this group did not develop PH or RVH. Two weeks post-MCT, E-dependent relaxation was impaired (ADP, 7 +/- 3% VS. c, 62 +/- 5%; A23187, 2 +/- 7% vs. C, 58 +/- 2%; ACh, 33 +/- 7% vs. C, 86 +/- 2%; P < 0.05) and ET-1 levels were elevated (1925 +/- 244 pg/g wwt vs. C, 469 +/- 59 pg/g wwt, P < 0.05), In addition, significant PH and RVH were present (PAP 33 +/- 4 mmHg vs. C 18 +/- 0.8 mmHg, P < 0.05; RVH index 0.40 +/- 0.006 vs. C, 0.25 +/- 0.01, P < 0.05). Incubation with 10 microM indomethacin, 150 U/ml superoxide dismutase or 300 microM L-arginine failed to restore impaired relaxation to ACh. In E-intact rings, relaxation to 10(-6) M glyceryl trinitrate (GTN) was inhibited at 1 week post-MCT (72 +/- 2% vs. C, 87 +/- 3%, P < 0.05) with further inhibition at 2 weeks (39 +/- 4%). Response to GTN in E-denuded rings was normal in MCT groups. CONCLUSIONS These results indicate that MCT injection in rats results in delayed but progressive endothelial injury and PH. Despite mild endothelial dysfunction 1 week post-MCT, NO-related relaxation and ET-1 levels are normal. At 2 weeks post-MCT, inhibition of E-dependent NO-related relaxation and elevation of ET-1 levels are associated with PH and RVH. Thus inhibition of NO production associated with elevated ET-1 levels may play an important role in the pathophysiology of MCT-induced PH.

Bilateral atrial appendectomy abolishes increased plasma atrial natriuretic peptide release and blunts sodium and water excretion during volume loading in conscious dogs.

The atrial appendages contain most of the atrial natriuretic factor (ANF) in the mammalian heart, and atrial appendage mechanical function predicts ANF secretion during volume loading. To demonstrate the crucial role of the atrial appendages in ANF release, we first measured hemodynamics and changes in plasma ANF after injection of 1,000 ml i.v. normal saline in conscious dogs and again after bilateral atrial appendectomy; we next measured changes in renal function using infusions of atriopeptin 24 to achieve plasma levels corresponding to levels achieved during volume loading; and we lastly measured renal function during acute volume expansion and also after atrial appendectomy. Plasma ANF increased from 65 +/- 11 to 246 +/- 54 pg/ml after volume loading but did not increase after atrial appendectomy. Atrial appendectomy did not alter the tachycardia or hemodynamic effects of volume loading. Infusion of 10 ng/kg/min atriopeptin 24 increased plasma ANF from 50 +/- 9 to 234 +/- 54 pg/ml, increased urine output 34 +/- 10%, and increased sodium excretion 62 +/- 10% in dogs with intact atrial appendages. Renal function was compared in dogs before atrial appendectomy: 20, 40, and 60 minutes after volume loading, urine flow rate increased by 5.9 +/- 0.5, 6.9 +/- 0.4, and 4.4 +/- 0.8 ml/min, while sodium excretion increased by 717 +/- 60, 839 +/- 84, and 582 +/- 57 mueq/min. After atrial appendectomy urine flow rate increased 2.1 +/- 0.7, 2.7 +/- 0.7, and 2.0 +/- 0.6 ml/min, and sodium excretion increased only by 327 +/- 110, 324 +/- 77, and 340 +/- 92 mueq/min (p less than 0.01) during volume loading.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional Renal Nitric Oxide Release in Stroke-Prone Spontaneously Hypertensive Rats

Diminished nitric oxide (NO) production has been implicated in the pathogenesis of salt-sensitive hypertension. We questioned whether such a defect is responsible for the malignant hypertension and nephrosclerosis in stroke-prone spontaneously hypertensive rats (SHRSP) fed a high-salt/stroke-prone diet (S) versus a regular diet (R). NO release from 30-minute incubates of cortex and outer and inner medulla were studied in SHRSP at 10, 12, and 16 weeks of age on the S diet versus R diet. SHRSP-S (n=16) exhibited a marked age-dependent increase in NO release, especially in the cortex. Increases were only modest in SHRSP-R (n=21). At 16 weeks, cortical NO was 93+/-25 versus 6+/-1 pmol/mg tissue in SHRSP-S versus SHRSP-R (P<.001). Immunohistochemical staining increased mostly for neuronal, slightly for endothelial, and negligibly for inducible isoforms of NO synthase and was predominantly in the cortex of SHRSP-S versus SHRSP-R. Despite similar hypertension in SHRSP-S versus SHRSP-R (mean arterial pressure, 174+/-7 versus 177+/-2 mm Hg), malignant nephrosclerosis was seen only in SHRSP-S, affecting 22+/-6% of glomeruli and 23+/-4 vessels per 100 glomeruli by 16 weeks. N omega-nitro-L-arginine (15 mg/kg per day) in SHRSP-S (n=6) abrogated the increase in cortical NO but further augmented the hypertension and accelerated lesion development. Wistar-Kyoto rats at 16 weeks on the R diet (n=8) had NO levels similar to those of SHRSP-R, showed increased cortical NO to only 28+/-10 pmol/mg on the S diet (n=9) (P<.05 versus SHRSP-S), but remained normotensive and lesion-free. We conclude that hypertension and lesion development in SHRSP are not due to deficient renal NO. Accelerated onset of malignant nephrosclerosis by NO synthase inhibition suggests that NO is protective in these animals, mitigating the effects of hypertension and S diet on renal pathology.

CHRONIC ALCOHOL INGESTION: EFFECTS ON WATER METABOLISM*

The diuretic action of alcohol has been recognized by the common man for many centuries. Scientific observations that established the diuretic action of ethanol as a specific phenomenon due to alcohol ingestion were made by Murray in 1932.’ In 1942, Eggleton2 showed that ethanol was probably acting on hypothalamic or pituitary levels, because pituitary extract inhibited the diuretic action of ethanol. This hypothesis has been confirmed by others,’+ and it is now well accepted that alcohol diuresis is due to a n inhibitory action on antidiuretic hormone release.’.S We have studied rats ingesting alcohol chronically (AC rats) that were maintained since birth on 12% (v/v) ethanol. They showed hypertrophy of the neurohypophysis, compared to normal rats (N rats). The enlarged neurohypophysis of AC rats also had a higher acid phosphatase content per milligram of tissue, which was interpreted as an effort by the pituicytes to facilitate vasopressin r e l e a ~ e . ~ We have continued our studies on the functional capacity of the neurohypophysial system and the sensitivity of the kidney in AC and N rats.

Variable arginine vasopressin levels in neonatal congestive heart failure

Arginine vasopressin levels in 17 neonates with cardiac disease were compared with control levels in 10 healthy newborn infants. Infants with congestive heart failure who were free of left ventricular outflow tract obstruction had a mean level of 80 +/- 18 pg/ml, which was significantly greater than the mean control level (p less than 0.001). Infants with congestive heart failure and left ventricular outflow tract obstruction had a mean vasopressin level of 3 +/- 0.7 pg/ml, which was lower than the mean control level of 6 +/- 0.7 pg/ml (p less than 0.05). The data suggest that impaired forward flow to high pressure sinoaortic and ventricular baroreceptors is necessary for vasopressin release in congestive heart failure. In left ventricular outflow tract obstruction with heart failure these receptors may be impaired or absent, leading to decreased vasopressin release. Low plasma arginine vasopressin may adversely affect circulatory homeostasis.

Dynamics of atrial peptide secretion in the coronary circulation of the conscious dog

The secretion of atrial natriuretic peptide by the heart is not simply the arterial-coronary sinus concentration difference times coronary blood flow, because only a small fraction of total coronary blood flow passes through the atria. We measured coronary sinus and arterial plasma atrial natriuretic factor (ANF) concentrations and blood flow to each part of the heart using the radioactive microsphere technique. Before acute volume expansion, the arterial-coronary sinus ANF difference was 305 +/- 23 pg/ml and rose to 1,009 +/- 220 pg/ml during volume expansion, whereas total coronary blood flow rose from 167 to 465 ml/min. Atrial blood flow rose from 2.9% to 4.6% of total coronary blood flow during volume expansion. ANF secretion rate increased from 51 to 469 ng/min. When divided by atrial weight, ANF secretion rate increased from 4.0 +/- 0.3 to 56 +/- 12 ng/min/g atrial tissue-in other words, from 0.3% to 3.7% of tissue ANF content each minute. Dividing by atrial blood flow indicated that the concentration of ANF leaving atrial tissue was 10,000 to 29,651 pg/ml, and the additional secretion of ANF was determined by the increase in coronary blood flow. Therefore, at least two mechanisms are responsible for altering coronary sinus ANF and circulating ANF: the release rate from atrial myocytes and the washout via changes in atrial blood flow.

Contribution of the ventricles and the atrial appendages to the elevation of plasma atrial natriuretic factor (ANF) during pacing-induced heart failure in conscious dogs

The goal of our study was to determine whether the elevation in plasma ANF was produced by the atrial appendages or ventricular tissue during pacing-induced heart failure in chronically instrumented conscious dogs (sham) or dogs with bilateral atrial appendectomy. Acute volume expansion caused a significant elevation of plasma ANF from 80±8 to 149±26 pg/ml (p<0.01) in sham dogs, but caused no significant change from 67±7 to 84±8 pg/ml in atrial appendectomized dogs. There were increases in left ventricular end-diastolic pressure (LVEDP) and left atrial pressure (LAP) in both groups of dogs. After rapid left ventricular pacing (210–240 beats/min) for 4 weeks to induce heart failure, dogs in both groups had tachycardia, elevated LVEDP and higher LAP. Plasma ANF was increased by 250% to 283±64 pg/ml (p<0.01) in sham dogs, and only 40% to 94±15 pg/ml (p>0.05) in atrial appendectomized dogs. In response to volume expansion, there were further increases in LVEDP and LAP in both groups of dogs, but plasma ANF was not elevated (288±39 pg/ml) in sham dogs and only slightly increased (132±7 pg/ml) in atrial appendectomized dogs. Our results suggest that, during pacing-induced heart failure, the atrial appendages are the major source of elevated plasma ANF, and the remaining atrial and ventricular tissue, even when maximally stretched, can only modestly increase plasma ANF.

Elevated arginine vasopressin and lowered atrial natriuretic factor associated with hypertension in coarctation of the Aorta

Impairment of humoral and neural regulation of blood pressure may contribute to preoperative and postoperative hypertension in coarctation of the aorta and may also affect the release of vasopressin and atrial natriuretic factor. Because vasopressin and atrial natriuretic factor have potent vasoactive effects, we measured plasma vasopressin and atrial natriuretic factor levels by radioimmunoassay before operation and for 5 days after operation in 11 patients aged 9 months to 12 years undergoing coarctation repair and in 12 control patients undergoing other cardiovascular operations. Six patients in the coarctation group required minimal antihypertensive therapy (group I) and five required prolonged intravenous antihypertensive therapy (group II). Before operation, vasopressin levels correlated with systolic blood pressure for all patients in the coarctation group (r = 0.83, p < 0.01) whereas atrial natriuretic factor levels did not. Before operation, atrial natriuretic factor levels were lower (28 +/- 5 vs 41 +/- 7 and 50 +/- 8 pg/ml, p < 0.05) and vasopressin levels were higher (28 +/- 6 vs 5.4 +/- 0.9 and 7 +/- 3 pg/ml, p < 0.05) in group II than in group I or control patients. Vasopressin levels were higher (p < 0.05) on the day of operation and on postoperative days 2 through 5 in group II than in group I and in control patients. Atrial natriuretic factor levels were lower during the day of operation in group II than in group I or in control patients (26 +/- 7 vs 51 +/- 16 and 50 +/- 7 pg/ml, p < 0.05) and remained lower than control values on postoperative days 1 and 3 through 5. Elevated vasopressin and lowered atrial natriuretic factor levels may contribute to preoperative and postoperative hypertension in coarctation.

Atrial natriuretic peptide replacement therapy in rats subjected to biatrial appendectomy

The postoperative fluid retention found in some patients after the Cox maze procedure has been attributed to surgically induced loss of atrial natriuretic peptide. We postulated that exogenous atrial natriuretic peptide could reverse this antidiuresis. A rat model was used to investigate this hypothesis. In group I, the sham group, the atrial appendages were left intact and the animals were then subjected to a fluid challenge equivalent to 1% of the animals body weight. In group II, after biatrial appendectomy, the animals were subjected to a fluid challenge similar to that in group I. Animals in group III underwent the same protocol as that for group II plus intravenous administration of atriopeptin III at varying concentrations. Urine output and plasma atrial natriuretic peptide levels were significantly decreased after biatrial appendectomies (p < or = 0.01). Urine output returned to control levels after biatrial appendectomies with low-dose atrial natriuretic peptide infusion (0.5 pmol/min = 25.5 pg/min), although circulating atrial natriuretic peptide levels were lower. Urine output and plasma atrial natriuretic peptide levels increased with atrial natriuretic peptide infusions between 0.5 and 50 pmol/min. Heart rate and mean blood pressure did not vary significantly with atrial natriuretic peptide infusions. Thus atrial natriuretic peptide can be used effectively in low doses to induce a diuresis after biatrial appendectomies. Atrial natriuretic peptide may have clinical application after the Cox maze procedure.