Atrial Natriuretic Peptide Attenuates Elevations in Ca2+ and Protects Hepatocytes by Stimulating Net Plasma Membrane Ca2+ Efflux

Abstract

Elevations in intracellular Ca2؉ concentration and calpain activity are common early events in cellular injury, including that of hepatocytes. Atrial natriuretic peptide is a circulating hormone that has been shown to be hepatoprotective. The aim of this study was to examine the effects of atrial natriuretic peptide on potentially harmful elevations in cytosolic free Ca 2؉ (24) that plays a key role in initiation of cellular injury and subsequent necrosis or apoptosis (6,(25)(26)(27)(28)(29)(30). In hepatocytes, calpain promotes hepatocyte plasma membrane blebbing (31), induces the mitochondrial permeability transition in hepatocyte necrosis (32), and contributes to hepatocyte necrosis in anoxia (33). Calpain has been implicated in hepatotoxicity in ischemia-reperfusion injury (34, 35), hemorrhagic shock (36), and bile acid toxicity (20).In the search for cytoprotective agents and therapeutic interventions, those which suppress injurious [Ca 2ϩ ] i rises are considered highly promising (2-6, 30). Natriuretic peptides attenuate elevations in cytosolic free Ca 2ϩ concentration ([Ca 2ϩ ] c ) in many cell types (37-50), including rat hepatocytes (51). Atrial natriuretic peptide (ANP) 3 is a circulating hormone released mainly by atrial myocytes (52) in response to stress conditions such as volume expansion or cardiac hypoxia. In addition to its hypotensive, vasodilatory, and natriuretic effects in the cardiovascular and renal systems (53), increasing evidence supports a more widespread role for ANP in several other organs (54), including the liver. ANP elevates cGMP in rat hepatocytes (51,(55)(56) through the guanylyl cyclase A receptor (57), and is cytoprotective in both isolated hepatocytes (51,55,58) and perfused liver (57, 59 -62). An early preliminary study showed that ANP attenuated both oxidant-induced cell injury and the accompanying [Ca 2ϩ ] i rise (51), suggesting that the hepatoprotective effects of ANP may be mediated by mechanisms involved in Ca 2ϩ homeostasis (51, 55, 57). We have since examined the effects of ANP on physiological alterations in [Ca 2ϩ ] c ; we showed that ANP, through protein kinase G (PKG), attenuates [Ca 2ϩ ] c oscillations, dramatically increases the basal rate of plasma membrane Ca 2ϩ efflux and modestly inhibits basal Ca 2ϩ influx in rat hepatocytes (56).

17β-Estradiol Elevates cGMP and, via Plasma Membrane Recruitment of Protein Kinase GIα, Stimulates Ca2+ Efflux from Rat Hepatocytes

“…17␤-Estradiol Stimulates PMCA-mediated Ca 2ϩ Efflux from Hepatocytes-Consistent with our previous findings (32)(33)(34)78), a basal rate of Ca 2ϩ efflux was recorded from intact rat hepatocytes in the absence of agonist (Fig. 4, A and G) and was blocked by caloxin 1b1, a specific PMCA inhibitor (79) (Fig.…”

“…In confirmation of our previous findings (33,34), immunoreactivity of the commercially available antibodies cGKI␣ (N-16) and cGKI␤ (L-16) (visualized using Alexa 594, red secondary) demonstrated expression of PKGI␣ and PKGI␤ in isolated rat hepatocytes (Fig. 3).…”

“…The specificity and efficacy of this inhibitor is demonstrated in Fig. 2A; NS2028 prevented any significant elevation of cGMP upon the addition of sodium nitroprusside, an NO donor that stimulates soluble guanylyl cyclase (21,32,33). In contrast, ANP, a known activator of particulate guanylyl cyclase (22,24,32), was still able to elevate cGMP to similar levels in the presence or absence of NS2028.…”

“…Fluorescence was measured at 37°C with constant stirring by using a PTI DeltaRam spectrofluorimeter (Photon Technologies International, Inc., Lawrenceville, NJ), exciting alternately at 340 and 380 nm and collecting emission at 510 nm. [Ca 2ϩ ] o was calculated by calibrating the photometric 340/380 nm ratio signal as described previously (33). ([Ca 2ϩ ] o ) never rose above 1.5 M, ensuring a true, unidirectional, Ca 2ϩ efflux measurement.…”

“…These include our own previous studies in rat hepatocytes; particulate guanylyl cyclase activation by ANP promotes plasma membrane recruitment of PKGI␣, Ca 2ϩ efflux stimulation, and [Ca 2ϩ ] c attenuation, whereas soluble guanylyl cyclase activators are without effect (32)(33)(34). Interestingly, both particulate guanylyl cyclase (4 -5, 7, 9, 14, 20) and soluble guanylyl cyclase (6,(35)(36)(37)(38) 44-2476-523701; E-mail: A.K.Green@warwick.ac.uk.…”