T.G. Flynn

Chronic hypertension in ANP knockout mice: contribution of peripheral resistance.

Atrial Natriuretic Peptide (ANP) exerts a chronic hypotensive effect which is mediated by a reduction in total peripheral resistance (TPR). Mice with a homozygous disruption of the pro-ANP gene (-/-) fail to synthesize ANP and develop chronic hypertension in comparison to their normotensive wild-type (+/+) siblings. In order to determine whether alterations in basal hemodynamics underlie the hypertension associated with lack of endogenous ANP activity, we used anesthetized mice to measure arterial blood pressure (ABP) and heart rate (HR), as well as cardiac output (CO) by thermodilution technique. -/- (n = 7) and +/+ (n = 10) mice of comparable weight and age were used. Stroke volume (SV) and TPR were derived from CO, HR, and ABP by a standard formula. ABP (mm Hg) was significantly higher in -/- (132+/-4) (P < 0.0001) than in +/+ mice (95+/-2). CO (ml min(-1)), HR(beats min(-1))and SV (microl beat(-1)) did not differ significantly between -/- and +/+ mice (CO -/- = 7.3+/-0.5, +/+ = 8.3+/-0.6; HR -/- = 407+/-22, +/+ = 462+/-21; SV -/- = 17.6+/-1.1, +/+ = 17.6+/-1.7). However, TPR (mm Hg ml(-1) min(-1)) was significantly elevated in -/- mice (18.4+/-0.7) compared to +/+ mice (12.3+/-1) (P = 0.0003). Autonomic ganglion blockade with a mixture of hexamethonium and pentolinium was followed by comparable percent reductions in CO (-/- = 28+/-4, +/+ = 29+/-3), HR (-/- = 9+/-4, +/+ = 16+/-4) and SV(-/- = 21+/-4, +/+ = 15+/-6) in both genotypes. However, the concomitant decrease in ABP (%) in -/- (41+/-2) was significantly greater than in +/+ (23+/-4) mice (P = 0.0009) and was accompanied by a significant reduction in TPR. We conclude that the hypertension associated with lack of endogenous ANP is due to elevated TPR, which is determined by an increase in cardiovascular autonomic tone.

Expression of B-type natriuretic peptide in atrial natriuretic peptide gene disrupted mice.

Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are two hormones produced and secreted by the heart to control blood pressure, body fluid homeostasis and electrolyte balance. Each peptide binds to a common family of 3 receptors (GC-A, GC-B and C-receptor) with varying degrees of affinity. The proANP gene disrupted mouse model provides an excellent opportunity to examine the regulation and expression of BNP in the absence of ANP. A new radioimmunoassay (RIA) was developed in order to measure mouse BNP peptide levels in the plasma, atrium and ventricle of the mouse. A detection limit of 3–6 pg/tube was achieved by this assay. Results show that plasma and ventricular level of BNP were unchanged among the three genotypes of mice. However, a significant decrease in the BNP level was noted in the atrium. The homozygous mutant (ANP–/–) had undetectable levels of BNP in the atrium, while the heterozygous (ANP+/–) and wild-type (ANP+/+) mice had 430 and 910 pg/mg in the atrium, respectively. Northern Blot analysis shows the ANP–/– mice has a 40% reduction of BNP mRNA level in the atrium and a 5-fold increase in the ventricle as compared with that of the ANP+/+ mouse. Our data suggest that there is a compensatory response of BNP expression to proANP gene disruption. Despite the changes in the atrial and ventricular tissue mRNA and peptide levels, the plasma BNP level remains unaltered in the ANP–/– mice. We conclude that the inability of BNP to completely compensate for the lack of ANP eventually leads to chronic hypertension in the proANP gene disrupted mice.

Immunoactive iso-ANP/BNP in plasma, tissues and atrial granules of the rat

We have developed a specific radioimmunoassay (RIA) for iso-rANP(1-45)/rBNP(51-95) using antiserum produced against a peptide comprise of the first 20 amino acid sequence of this cardiac hormone. Using this RIA, we found that atria contained the highest amount of iso-rANP(1-45) (567.37 pmol/g) which is about 140-fold higher than ventricles (4.32 pmol/g). With the exception of the lung and kidney, all other tissues had negligible amounts. The plasma level was 1.4 fmol/ml and the only molecular form found was the 45 amino acid form. HPLC analysis of extracts of isolated, purified atrial granules revealed that, like atrial natriuretic peptide (ANP), iso-rANP/rBNP is also stored in these granules. However, while ANP is stored predominantly as pro-ANP(1-126) and cleaved during or after release, iso-ANP/BNP is stored as the 45 amino acid form and may be processed prior to storage in the granules.

Lack of biologic activity or specific binding of amino-terminal pro-ANP segments in the rat.

In addition to atrial natriuretic peptide (ANP99-126) itself, linear peptide fragments from its N-terminal prohormone segment (pro-ANP) have been reported to have biological activity. In vivo, diuresis and natriuresis, as well as hypotension have been observed. In vitro, sodium uptake into medullary collecting duct cells was inhibited, and tone of vascular smooth muscle was reduced, associated with activation of guanylate cyclase. Such previous studies have used heterologous peptides and species, e.g., human pro-ANP1-30 or pro-ANP31-67, tested in rat, pig, or dog. The present experiments were designed to test whether rat pro-ANP1-30 or pro-ANP31-67 were natriuretic and hypotensive in rats, whether the two peptides showed specific binding to plasma membranes from rat kidney cortex or aorta, and whether they affected particulate guanylate cyclase activity in rat glomerular membranes. To extend in vitro results from the literature, the effect of human pro-ANP31-67 on transport in the rat medullary collecting duct in vivo was also tested. Although rat ANP99-126, as expected, increased diuresis and natriuresis, associated with inhibition of transport in the medullary collecting duct, in identically treated rats human pro-ANP31-67 was without effect. Similarly, only the ANP99-126 infusion resulted in reduction of arterial blood pressure. Furthermore, no diuretic, natriuretic, or hypotensive responses were observed in rats infused with either rat pro-ANP31-67 or pro-ANP1-30. In plasma membranes from rat kidney cortex or aorta, neither of the rat prosegments showed specific binding, or interference with ANP99-126 receptors. Finally, in contrast to ANP99-126, neither of the prosegments was able to increase basal guanylate cyclase activity in rat glomerular membranes. Therefore, under our experimental conditions we were unable to replicate the earlier results. This study thus does not support a regulatory role for pro-ANP fragments in blood volume or blood pressure homeostasis.

Radioimmunoassay for Rat B-Type Natriuretic Peptide (BNP-45)

Rat BNP-45 is the main circulating form of BNP in rat plasma. To understand the role of BNP in physiological and pathophysiological conditions, a specific radioimmunoassay (RIA) for the quantitative determination of the peptide in plasma and tissues is necessary. An assay using rBNP-45 as the standard in conjunction with antisera directed against this peptide has not been described in the literature, though some investigators have reported values ranging from 0.73-2.0 pmol/L using either BNP-26 or BNP-32 as the standard peptide. Unfortunately, these forms of BNP do not exist in rat plasma. In our studies, we have developed a specific RIA for rBNP-45 using rBNP-45 as the standard peptide and Tyro-rBNP-45 as the radioligand. We have used two specific antisera for assay purposes; one against rBNP-45, and the second to a peptide composed of the first 20 amino acids of rBNP-45 (rBNP[1-20]). The recovery of various amounts of rBNP-45 added to control plasma was 50-80% depending on the method of extraction and purification. The interassay and intraassay coefficients of variation were 12% and 6% respectively. Values obtained were similar for blood sampled by either cardiac puncture, decapitation, or aortic puncture. The method was used to measure rBNP-45 in the plasma of normal (WKY) and Spontaneously Hypertensive (SHR) rats. The values obtained were 5.46 +/- 0.43 and 19.6 +/- 2.36 pmol/L respectively. The rat atrial natriuretic peptide (ANP[99-126]) values in the same extracts were 23.2 +/- 0.45 and 51.6 +/- 3.16 pmol/L.

Plasma clearance and tissue binding of rANP[99–126] and iso-rANP[1–45] in the rat

Plasma clearance and tissue binding of atrial natriuretic peptide (ANP) and iso-ANP were compared in Inactin-anaesthetized rats. It was found that the plasma half-life of iso-ANP was comparable to ANP. Appearance of trichloroacetic acid-soluble radioactivity of iso-ANP in the plasma was considerably slower than that of ANP, suggesting that the metabolic process of these two peptides may be different. Although the binding distribution of these two peptides was similar, the total binding of iso-ANP to organs other than the kidney was much lower. The kidney, lung, heart and adrenal gland appeared to be major target organs for iso-ANP. Autoradiography showed that iso-ANP bound specifically to the renal glomerulus and proximal part of the proximal tubule. This latter binding site in the kidney was not apparent with ANP, suggesting that iso-ANP may exerts its physiological action at different sites in this organ.

Measurement of prolactin release and cytosolic calcium in estradiol-primed lactotrophs

We have developed a perifusion system that can measure both changes of cytosolic free calcium concentration [Ca2+]i and prolactin release simultaneously from cultured lactotrophs. This model incorporated a commonly-used perifusion system to a spectrofluorometer. Indo-1 loaded cells were injected into Sephadex G-150 matrix in the cuvette at a site where the emitting light of the fluorometer projects. During perifusion periods, the perifusate was collected in a fraction collector, while optical density of the emitting light at 405 nm was recorded. The [Ca2+]i was calculated based on an ionomycin and Mn2+ quenching technique. As expected, TRH (1 mumol/l) stimulated prolactin release from cultured lactotrophs in this system. We further observed that prolactin releases as induced by TRH and ionomycin were not proportional with changes of the [Ca2+]i, suggesting that changes of [Ca2+]i is not the sole final pathway of intracellular transduction systems for prolactin release.

Circulatory and metabolic responses in awake dogs to infusion of iso-rANP/(rBNP)

We reported that a second rat atrial peptide, iso-atrial natriuretic peptide (iso-rANP(1-45)) and a potential putative homologue, iso-rANP(17-45) (identical with rat brain natriuretic peptide except for one amino acid) elicited circulatory and renal responses in anesthetized rats. In the present studies, low-dose intravenous infusions of iso-rANP(1-45) (6.3-25 pmol kg-1 min-1) and iso-rANP(17-45) (12.5-50 pmol kg-1 min-1) into conscious dogs produced subtle circulatory effects compared to control studies. Relative to oxygen consumption, cardiac output was lower and total peripheral resistance higher with both iso-rANP(1-45) and iso-rANP(17-45). Heart rate tended to be slightly lower relative to control studies during peptide infusions, and the highest infusion doses caused a decrease in mean arterial pressure. Plasma protein increased and plasma osmolality decreased with iso-rANP(1-45); infusion of iso-rANP(17-45) caused a decrease in the respiratory exchange ratio. The mechanism of action of iso-rANP may have been direct, via an active receptor. However, we previously reported for these same experiments that infusion of iso-rANP(1-45) and iso-rANP(17-45) increased plasma ANP and decreased plasma renin activity. Thus, circulatory changes during infusion of iso-rANP were consistent with an indirect mechanism related to increased endogenous ANP.