Erwin J. Landon

A novel angiotensin II type 2 receptor signaling pathway: possible role in cardiac hypertrophy

We describe a novel signaling mechanism mediated by the G‐protein‐coupled receptor (GPCR) angiotensin II (Ang II) type 2 receptor (AT2). Yeast two‐hybrid studies and affinity column binding assay show that the isolated AT2 C‐terminus binds to the transcription factor promyelocytic zinc finger protein (PLZF). Cellular studies employing confocal microscopy show that Ang II stimulation induces cytosolic PLZF to co‐localize with AT2 at the plasma membrane, then drives AT2 and PLZF to internalize. PLZF slowly emerges in the nucleus whereas AT2 accumulates in the perinuclear region. Nuclear PLZF binds to a consensus sequence of the phosphatidylinositol‐3 kinase p85α subunit (p85α PI3K) gene. AT2 enhances expression of p85α PI3K followed by enhanced p70S6 kinase, essential to protein synthesis. An inactive mutant of PLZF abolishes this effect. PLZF is expressed robustly in the heart in contrast to many other tissues. This cardiac selective pathway involving AT2, PLZF and p85α PI3K may explain the absence of a cardiac hypertrophic response in AT2 gene‐deleted mice.

Calcium pump activity of the renal plasma membrane and renal microsomes

Abstract ATP-dependent Ca2+ uptake distinct from that of the mitochondria is found in both plasma membrane and microsomal membranes of rat kidney. Activity attributed to these fractions is enhanced by ammonium oxalate and is apparently insensitive to NaN3. In contrast, rat kidney mitochondrial Ca2+ uptake is blocked by NaN3. The pH of optimal activity is significantly higher for the mitochondrial fraction. Microsomal membrane Ca2+ uptake differs from that of the plasma membrane. Microsomal membranes are four times as active as the plasma membrane at high (5 mM) ATP levels. Apparent K m values for Mg2+-ATP differ in the two preparations with a higher affinity for Mg2+-ATP found in the plasma membrane Ca2+ uptake activity of the plasma membrane preparation is readily inhibited by Na+. Sucrose gradient density fractionation indicates that the observed microsomal membrane Ca2+ pump activity is associated with membrane vesicles derived from the endoplasmic reticulum. Ca2+ pump activity of both plasma membrane and microsomal fraction is depressed din the adrenalectomized rat. This activity is not restored by a single natriuretic dose of aldosterone.

A Calcium Pump in Vascular Smooth Muscle

A microsomal cell fraction derived from the intimal-medial layer of rabbit aorta takes up calcium in the presence of magnesium and adenosine triphosphate. The rate of uptake of calcium is slower than that observed in skeletal muscle microsomes. Uptake of calcium by mitochondria from the aorta is even more limited and, unlike microsomal uptake, is inhibited by azide.

Effects of calcium channel blocking agents on calcium and centrilobular necrosis in the liver of rats treated with hepatotoxic agents

Carbon tetrachloride, chloroform, dimethylnitrosamine, thioacetamide or acetaminophen was each administered to rats in a single hepatotoxic dose. Nifedipine, verapamil or chlorpromazine was administered in association with the hepatotoxic agents to determine if calcium channel blocking agents would prevent an increase in liver cell calcium associated with hepatotoxicity and to determine if these agents would protect against the development of centrilobular necrosis. Following a latent period different for each toxic agent, a 4- to 18-fold increase in liver cell calcium content had occurred by 24 hr. The calcium increase and the centrilobular necrosis (mean histologic score) were correlated. A relatively high calcium to necrosis ratio was obtained with dimethylnitrosamine, thioacetamide and acetaminophen. A lesser calcium to necrosis ratio was obtained with chloroform and carbon tetrachloride, the two toxic agents that destroyed the intracellular calcium sequestration activity of the liver endoplasmic reticulum. Nifedipine or chlorpromazine, administered prior to and 7 hr after the toxic agent, completely prevented the centrilobular necrosis caused by thioacetamide, carbon tetrachloride and acetaminophen; almost completely prevented necrosis with dimethylnitrosamine; and provided partial protection against chloroform toxicity. Two doses of verapamil provided partial protection against necrosis when carbon tetrachloride was the toxic agent and provided almost complete protection with dimethylnitrosamine. A reduction in liver cell calcium was associated with the protective action of the three calcium channel blocking agents. These findings are compared with earlier studies of the protective effects of calcium channel blocking agents in cardiac ischemia.

Localization of Calcium Pump Activity in Smooth Muscle

A microsomal fraction isolated from longitudinal smooth muscle of guinea pig ileum actively sequesters calcium ion in the presence of magnesium and adenosine triphosphate in a fashion previously described for microsomes of the rabbit aorta. This activity in guinea pig ileum appears to be associated primarily with the plasma membrane as is found in the red cell. By contrast the uptake of calcium in aortic smooth muscle appears to be associated to an appreciable extent with intracellular membranes, possibly analogous to the sarcoplasmic reticulum of skeletal muscle.

Sodium- and potassium-dependent adenosine triphosphatase activity in a rat-kidney endoplasmic reticulum fraction.

Abstract ATPase activity has been characterized in a rat-kidney microsomal membrane preparation. Dialyzed preparations have a magnesium-dependent ATPase that is activated by sodium and potassium added in combination. Evidence is presented for separate sites of binding of sodium and potassium in the preparation. This sodium-potassium-dependent ATPase activity is inhibited by ouabain, organic mercurial diuretics and calcium. Ouabain inhibition appears to be on the uptake of cations rather than the ATPase activity. Hydrogen-ion effects on ATPase activity are examined and discussed. It is postulated that the activity observed is a component of the active-transport system for sodium and potassium in the rat kidney.

Energy-dependent calcium uptake activity of microsomes from the aorta of normal and hypertensive rats

Energy-dependent calcium uptake activity of microsomes isolated from the rat aorta has been characterized. The microsomes consist of smooth membrane vesicles which in the presence of MG-ATP as an energy source continuously sequester calcium over a 60-min period. This calcium uptake is greatly stimulated by oxalate anion which serves as a calcium trapping agent. Unlike the calcium uptake of mitochondria this uptake is not inhibited by sodium azide. Sucrose density gradient analysis of the microsomal calcium uptake suggests that the system is associated with the sarcoplasmic reticulum. In presence of 5 mM Mg-ATP and 20 muM calcium approximately 38 nmol of calcium per mg of microsomal protein are taken up in 20 min. In the absence of ATP, less than 2 nmol of calcium per mg of protein are taken up in the first 2 min with no further uptake of calcium in subsequent time periods. When calcium uptake activity is plotted against calcium or ATP concentration of the medium, half maximal activity is calculated for 24.3 muM calcium and for 1.6 mM ATP. The calcium uptake characteristics of the rat aorta microsomes are compatible with a postulated role in the relaxation of the vascular smooth muscle and the provision of an intracellular calcium store for muscle contraction. Aorta microsomes from SHR rats (a genetic strain that is spontaneously hypertensive) have a significantly reduced uptake when compared with the corresponding nonhypertensive control strain. The level of calcium and ATP for half maximal activity of the rat aorta microsomal calcium uptake system is approximately the same in the SHR and the control strain. The rate of release of calcium from rat aorta microsomes is apparently identical in SHR strain and control. The calcium uptake activity of kidney and liver microsomes isolated from the SHR strain and control. The calcium uptake activity of kidney and liver microsomes isolated from the SHR rat appears to be identical to that found in the control strain.

The calcium accumulation in a microsomal fraction from porcine coronary artery smooth muscle. A study of the heterogeneity of the fraction.

1. Microsomes prepared from the combined media and intima of pig coronary artery, take up Ca in an ATP-dependent way. This uptake is stimulated by oxalate. 2. Conditions have been determined to optimize the preparation of the microsomes in terms of their Ca accumulation activity. Careful homogenization of the tissue mince in 0.25 M sucrose by means of a Potter-Elvehjem homogenizer gives microsomal preparations with the highest specific activity for Ca accumulation. 3. Arguments are presented to support the hypothesis that, even in the absence of oxalate, Ca accumulation occurs into the lumen of the vesicles, and that these vesicles have a low Ca permeability. 4. Density gradient analysis shows that the microsomal fraction prepared from pig coronary artery media and intima is composed of vesicles that are heterogeneous in enzymatic composition. 5. Adenylate cyclase appears to be a predominantly plasma membrane-bound enzyme. Rotenone-insensitive NADH-cytochrome c reductase and choline phosphotransferase, two putative markers for internal membranes, give distinct banding patterns on on isopycnic centrifugation, indicating different intracellular localization. 6. There is a difference between the density gradient distribution pattern of Ca uptake measured in the presence or absence of oxalate. The latter coincides more closely with plasma membrane markers. The former resembles more the distribution of rotenone-insensitive NADH-cytochrome c reductase.

Inhibition of the cardiac mitochondrial calcium pump by adriamycin in vitro

Abstract Adriamycin and daunomycin are clinically useful antineoplastic agents, known for their ability to produce a delayed cardiomyopathy upon chronic administration. Recently it has been suggested that an alteration of myocardial calcium metabolism precedes the cardiomyopathy. This study demonstrates that these cardiotoxic antibiotics inhibit one of the subcellular systems thought to be important in the regulation of calcium metabolism in the heart. In the present communication we report that calcium translocation by cardiac mitochondria is inhibited by these anthracyclines, but that calcium translocation by the cardiac sarcoplasmic reticulum is not inhibited.

Antagonism or Synergism ROLE OF TYROSINE PHOSPHATASES SHP-1 AND SHP-2 IN GROWTH FACTOR SIGNALING

SHP-1 and SHP-2 are two Src homology 2 domain-containing tyrosine phosphatases with major pathological implications in cell growth regulating signaling. They share significant overall sequence identity, but their biological functions are often opposite. SHP-1 is generally considered as a negative signal transducer and SHP-2 as a positive one. However, the precise role of each enzyme in shared signaling pathways is not well defined. In this study, we investigated the interaction of these two enzymes in a single cell system by knocking down their expressions with small interfering RNAs and analyzing the effects on epidermal growth factor signaling. Interestingly, knockdown of either SHP-1 or SHP-2 caused significant reduction in the activation of ERK1/2 but not Akt. Furthermore, SHP-1, SHP-2, and Gab1 formed a signaling complex, and SHP-1 and SHP-2 interact with each other. The interaction of SHP-1 with Gab1 is mediated by SHP-2 because it was abrogated by knockdown of SHP-2, and SHP-2, but not SHP-1, binds directly to tyrosine-phosphorylated Gab1. Together, the data revealed that both SHP-1 and SHP-2 have a positive role in epidermal growth factor-induced ERK1/2 activation and that they act cooperatively rather than antagonistically. The interaction of SHP-1 and SHP-2 may be responsible for previously unexpected novel regulatory mechanism of cell signaling by tyrosine phosphatases.