Amritlal Mandal

• Ph.D.
• Researcher at University of Arizona

Previously we showed hyperosmotic solution caused TRPV1‐dependent NKCC1 activation in the lens by a mechanism that involved ERK1/2 signaling. In various tissues, integrins and the cytoskeletal network play a role in responses to osmotic stress. Here, we examined the association between integrins and TRPV1‐dependent activation of NKCC1 in mouse lens...

Endothelin-1 (ET-1) is a potent endogenously derived vasoconstrictor, which increases pulmonary hypertension via stimulation of [Ca2+]i level in pulmonary artery smooth muscle cells (PASMCs). In this communication, we sought to investigate the mechanism by which ET-1 causes stimulation of Ca2+ concentration in caveolae vesicles of bovine PASMCs (BP...

Chronic obstructive pulmonary disease (COPD) is a lung disease that is often associated with chronic bronchitis, bronchiolitis and emphysema. The disease pathology is heterogeneous in nature and usually results from several environmental factors including cigarette smoke, biomass smoke particle, diesel and automobile exhausts that can potentially e...

Experimental protocol • Primary cultured pig lens epithelium cell (p3) exposed to 400mM hyperosmotic solution for 0, 2 and 5 min were used to test the efficiency of the supplied blocking buffers. • Cell lysate (RIPA, pH 7.4) used (20µg protein/lane). • Dual-wavelength Chameleon prestained protein standard ladder (Li-Cor# 978-16526, 1µl/lane) was...

Lens ion homeostasis is crucial in maintaining water content and, in turn, refractive index and transparency of the multicellular syncytium-like structure. New information is emerging on the regulation of ion transport in the lens by mechanisms that rely on Transient Receptor Potential Vanilloid (TRPV) ion channels. We found recently that TRPV1 act...

Recently we determined that the Transient Receptor Potential Vanilloid 4 ion channel (TRPV4) has a crucial signaling role in a pathway that regulates various aspects of lens epithelium function. Here, we report on a different TRPV channel, TRPV1, in porcine lens. The presence of TRPV1 in the lens was evident from RT-PCR studies and Western blot ana...

Regulation of blood pressure (BP) depends mostly on genetic and environmental factors. Among different physiological mechanisms responsible for a discernible increase in BP (hypertension), two major mechanisms are important: (a) matrix remodeling associated with arterial wall thickening with apparent reduction in blood flow and (b) activation of re...

Herein, synthesis of a Dnp-labeled peptide, Dnp-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-CONH2, is described by the Fmoc solid-phase method. Post-synthesis of the peptide was purified by reversed-phase HPLC. The purity of the peptide was determined by nuclear magnetic resonance total correlation spectroscopy (NMR TOCSY), and the validity of the peptide as...

Purpose: Na,K-ATPase activity in lens epithelium is subject to control by Src family tyrosine kinases (SFKs). Previously we showed hyposmotic solution causes an SFK-dependent increase in Na,K-ATPase activity in the epithelium. Here we explored the role of cAMP in the signaling mechanism responsible for the SFK and Na,K-ATPase response. Methods:...

Maintenance of cytoplasmic calcium ([Ca2+]i) at physiological level is a constant challenge for every cell including endothelial and smooth muscle cells of pulmonary vasculature. The cells are equipped with complex regulatory mechanisms to deal with a relatively large Ca2+ gradient that exists not only between intracellular and extracellular spaces...

Na+/K+-ATPase (NKA) plays the key role in maintaining Na+ and K+ gradients in cells, which is essential for regulation of cell volume and membrane potential. PLM (aka FXYD1) interacts with NKA and Na+/Ca2+ exchanger (NCX) and modulates their activities in tissue specific and physiological state specific manner. Protein kinase A (PKA) and protein ki...

Na+/K+-ATPase (NKA), a transmembrane protein, facilitates active transport of three Na+ out of the cell and two K+ into the cell with the expense of an ATP. It plays an important role in regulating the ionic homeostasis and maintaining membrane potential. Additionally, NKA plays a crucial role in driving a variety of secondary transport processes s...

Insulin has been shown to elicit changes of Na,K-ATPase activity in various tissues. Na,K-ATPase in the nonpigmented ciliary epithelium (NPE) plays a role in aqueous humor secretion and changes of Na,K-ATPase activity impact the driving force. Because we detect a change of NPE Na,K-ATPase activity in response to insulin, studies were carried out to...

To function normally, all cells must maintain ion homeostasis, establish a membrane potential, and regulate water content. These actions require active Na-K transport provided by Na,K-ATPase. The lens, however, is made up almost entirely of fiber cells that have little or no Na,K-ATPase activity. Lens ion and water homeostasis rely on Na,K-ATPase a...

The bulk of the lens consists of tightly packed fiber cells. Because mature lens fibers lack mitochondria and other organelles, lens homeostasis relies on a monolayer of epithelial cells at the anterior surface. The detection of various signaling pathways in lens epithelial cells suggests they respond to stimuli that influence lens function. Focusi...

Aneurysms develop as a result of chronic inflammation of vascular bed, where progressive destruction of structural proteins, especially elastin and collagen of smooth muscle cells have been shown to manifest. The underlying mechanisms are an increase in local production of proinflammatory cytokines and subsequent increase in proteases, especially m...

Aneurysms develop as a result of chronic inflammation of vascular bed, where progressive destruction of structural proteins, especially elastin and collagen of smooth muscle cells have been shown to manifest. The underlying mechanisms are an increase in local production of proinflammatory cytokines and subsequent increase in proteases, especially m...

Aneurysms develop as a result of chronic inflammation of vascular bed, where progressive destruction of structural proteins, especially elastin and collagen of smooth muscle cells have been shown to manifest. The underlying mechanisms are an increase in local production of proinflammatory cytokines and subsequent increase in proteases, especially m...

The nitric oxide (NO) donor sodium nitroprusside (SNP) is known to reduce aqueous humor (AH) secretion in the isolated porcine eye. Previously, SNP was found to inhibit Na,K-ATPase activity in nonpigmented ciliary epithelium (NPE), AH-secreting cells, through a cGMP/protein kinase G (PKG)-mediated pathway. Here we show Src family kinase (SFK) activ...

Aneurysms develop as a result of chronic inflammation of vascular bed, where progressive destruction of structural proteins, especially elastin and collagen of smooth muscle cells have been shown to manifest. The underlying mechanisms are an increase in local production of proinflammatory cytokines and subsequent increase in proteases, especially m...

Aneurysms develop as a result of chronic inflammation of vascular bed, where progressive destruction of structural proteins, especially elastin and collagen of smooth muscle cells have been shown to manifest. The underlying mechanisms are an increase in local production of proinflammatory cytokines and subsequent increase in proteases, especially m...

Aneurysms develop as a result of chronic inflammation of vascular bed, where progressive destruction of structural proteins, especially elastin and collagen of smooth muscle cells have been shown to manifest. The underlying mechanisms are an increase in local production of proinflammatory cytokines and subsequent increase in proteases, especially m...

Purpose: The nonpigmented ciliary epithelium (NPE) is rich in soluble adenylyl cyclase (sAC), a proposed cytoplasmic bicarbonate sensor. Here, we examine the contribution of sAC to an increase in cyclic AMP (cAMP) and changes in a key ion transporter, H(+)-ATPase, in NPE exposed to acetazolamide, a carbonic anhydrase inhibitor (CAI). Methods: Cy...

The nitric oxide (NO) donor SNP reduces aqueous humor (AH) secretion in the isolated porcine eye. SNP inhibits Na,K‐ATPase activity in AH‐secreting tissue, nonpigmented ciliary epithelium (NPE), through a cGMP/PKG‐mediated pathway. Here we study the role of Src family kinases (SFKs) in the Na,K‐ATPase activity response to NO. NPE was cultured and N...

We have recently reported that treatment of bovine pulmonary artery smooth muscle cells with the thromboxane A(2) mimetic, U46619 stimulated NADPH oxidase derived O(2)(·-) level, which subsequently caused marked increase in [Ca(2+)](i)[17]. Herein, we demonstrated that O(2)(·-)-mediated increase in [Ca(2+)](i) stimulates an aprotinin sensitive prot...

In several tissues, transient receptor potential vanilloid 4 (TRPV4) channels are involved in the response to hyposmotic challenge. Here we report TRPV4 protein in porcine lens epithelium and show that TRPV4 activation is an important step in the response of the lens to hyposmotic stress. Hyposmotic solution (200 mosM) elicited ATP release from int...

In several tissues TRPV4 channels are involved in the response to hyposmotic challenge. Our objective was to detect TRPV4 protein in porcine lens epithelium and its role in hyposmotic stress‐induced ATP release. The Western blot analysis showed presence of TRPV4 in the lens epithelial homogenate. The TRPV4 antagonist RN 1734 (10μM) completely preve...

Aqueous humor secretion is the result of net ion transport that involves the coordinated operation of Na,K‐ATPase in the nonpigmented ciliary epithelium (NPE) with several other basolateral transporters and channels in the ciliary epithelium bilayer. Here, we report on the ability of DIDS to reduce aqueous humor secretion and present studies to exa...

Many cells respond to osmotic stress by releasing ATP that acts as an extracellular signal for purinergic receptors. Receptor‐triggered increases of calcium and activation of Src family tyrosine kinases (SFKs) have previously been observed in lens epithelium. Here we examined the effect of hyposmotic stress on cytoplasmic calcium [(Ca ²⁺ ) i ] resp...

Purinergic receptors in lens epithelium suggest lens function can be altered by chemical signals from aqueous humor or the lens itself. Here we show release of ATP by intact porcine lenses exposed to hyposmotic solution (200 mOsm). 18α-glycyrrhetinic acid (AGA) added together with probenecid eliminated the ATP increase. N-ethylmaleimide (200 µM), a...

Previous studies show Src family kinase (SFK) activation is involved in a response that stimulates Na,K-ATPase. Here, we tested whether SFK activation is involved in the Na,K-ATPase response to endothelin-1 (ET-1). Intact porcine lenses were exposed to 100 nM ET-1 for 5-30 min. Then, the epithelium was removed and used for Na,K-ATPase activity meas...

Purpose Purinergic receptors in the lens suggest function can be altered by agonists present in aqueous humor. Some agonists may originate from the lens itself. Here, we examine the ability of osmotic shock to trigger ATP release from the intact lens. Methods Porcine lenses were exposed to hyposmotic (200 mOsm) or hyperosmotic (500mOsm) solution an...

NHE1, NHE2 and NHE3 mRNA detected in CP-A cells and JHEsoAD1 cells. mRNA levels were measured by RT-PCR from mRNA obtained from three independent experiments (*p<0.05 compared to CP-A cells). (TIF)

Lysosomal damage accompanies DCA-mediated intracellular acidification. A) Representative fluorescent microscopy images showing Lysotracker-Red in CP-A cells following 60 minutes treatment with or without 0.4 mM DCA in the presence or absence of 20 uM EIPA. Red signal indicates acidic vesicles, blue signal is nuclear counterstain (DAPI). B) Represen...

DCA induced ATP depletion in JHEsoAD1 cells. The cells were exposed for 2 hours to various concentration of DCA in the presence or absence of EIPA and ATP levels were measured by Enliten ATP Assay System Bioluminiscence Kit according the manufacturer's instructions. EIPA prevents ATP depletion (*p<0.05 compared to control). (TIF)

Zoniporide prevents DCA-induced cell death. The graph shows data from MTS assay (n = 4) in JHEsoAd1 cells detected 24 hours following a 120 minute exposure to 0.4 mM DCA in the presence or absence of 20 mM zoniporide (*p<0.05). (TIF)

PKC inhibition does not prevent changes in intracellular Na+ and K+. in JHEsoAd1 cells treated with DCA. JHEsoAd 1 cells were pretreated for 30 minutes with 10 mM Go6983 and then exposed to 0.4 mM DCA for 60 minutes in the presence or absence of Go6983 (n = 3; *p<0.05 compared to control). (TIF)

Inhibition of Na+ influx with EIPA prevents DCA-induced cell death in CP-A cells. A) Representative contrast microscopy images of CP-A cells following 120 minute incubation with and without 0.4 mM DCA in the presence or absence of 20 uM EIPA. Yellow arrows indicate damaged and apoptotic cells. B) Caspase-3/7 activity (n = 4) measured 24 hours follo...

Apoptosis resistance is a hallmark of cancer cells. Typically, bile acids induce apoptosis. However during gastrointestinal (GI) tumorigenesis the cancer cells develop resistance to bile acid-induced cell death. To understand how bile acids induce apoptosis resistance we first need to identify the molecular pathways that initiate apoptosis in respo...

Purpose Earlier studies point to the involvement of Src family tyrosine kinases (SFKs) in the stimulation of of Na,K‐ATPase activity by purinergic receptor agonists ATP and UTP. Src itself was activated (Tamiya, et al. 2007, Am. J. Physiol. 293: C790‐6). Here, we examined the role of SFKs in the response to endothelin‐1 (ET‐1), an ET receptor agoni...

Recent investigations demonstrate increased Na/H exchanger-1 (NHE-1) activity and plasma levels of ouabain-like factor in spontaneously hypertensive rats. At nanomolar concentrations, ouabain increases Na-K-ATPase activity, induces cell proliferation, and activates complex signaling cascades. We hypothesize that the activity of NHE-1 and Na-K-ATPas...

Elevated intraocular pressure is associated with glaucomatous optic nerve damage. Other investigators have shown functional changes in optic nerve head astrocytes subjected to elevated hydrostatic pressure (HP) for 1 to 5 days. Recently, the authors reported ERK1/2, p90(RSK) and NHE1 phosphorylation after 2 hours. Here they examine calcium response...

To better understand how nitric oxide (NO) alters the function of the nonpigmented ciliary epithelium (NPE), studies were performed to determine the influence of NO on sodium-hydrogen exchanger (NHE) activity. Cytoplasmic pH (pH(i)) was measured in cultured porcine NPE loaded with BCECF (2',7'-bis(2-carboxyl)-5(6)-carboxyfluorescein-acetoxyethyl es...

Optic nerve head astrocytes become abnormal in eyes that have elevated intraocular pressure, and cultured astrocytes display altered protein expression after being subjected for > or = 1 days to elevated hydrostatic pressure. Here we show that 2-h elevated hydrostatic pressure (15 or 30 mmHg) causes phosphorylation of ERK1/2, ribosomal S6 protein k...

Objective Optic nerve head (ONH) astrocytes are thought to involve in the degeneration of ganglion cells and remodeling of surrounding extracellular matrix in glaucoma. Elevated intraocular pressure (IOP) is the main glaucoma risk factor. Here we report altered sodium hydrogen exchange (NHE) activity in cells exposed to elevated hydrostatic pressur...

Plasma cardioglycosides (ouabain) levels increase in rat models of hypertension and in humans with heart and kidney failure. Ouabain activates Src, ERK, and Akt, resulting in increased 86Rb uptake and cell proliferation. NHE1 is also activated by Akt, suggesting coordinate regulation of NHE1 and Na‐K ATPase (Na‐K). We hypothesize that ouabain regul...

We sought to determine the mechanisms of an increase in Ca(2+) level in caveolae vesicles in pulmonary smooth muscle plasma membrane during Na(+)/K(+)-ATPase inhibition by ouabain. The caveolae vesicles isolated by density gradient centrifugation were characterized by electron microscopic and immunologic studies and determined ouabain induced incre...

Sodium-dependent transporters are inhibited indirectly by the Na-K-ATPase inhibitor ouabain. Here we report stimulation of sodium-hydrogen exchange (NHE) in ouabain-treated cells. BCECF was used to measure cytoplasmic pH in cultured rat optic nerve astrocytes. Ammonium chloride was applied to acid load the cells. On removal of ammonium chloride, cy...

Objective: Earlier we showed that 1 μM ouabain increased capacitative calcium entry (CCE) but did not increase cell sodium1. Here we studied cytoplasmic pH recovery after an acid load in ouabain‐treated cells and tested whether pH response were linked to altered calcium entry. Methods: Ammonium chloride (20mM) was used to acid load the cells. BCECF...

The properties of Ca(2+)-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C(12)E(8)) and Triton X-100 as the solubilizing agents. Solubilization with DHP...

Treatment of bovine pulmonary artery smooth muscle with the O2•− generating system hypoxanthine plus xanthine oxidase stimulated MMP-2 activity and PKC activity; and inhibited Na+ dependent Ca2+ uptake in the microsomes. Pretreatment of the smooth muscle with SOD (the O2•− scavenger) and TIMP-2 (MMP-2 inhibitor) prevented the increase in MMP-2 acti...

Treatment of microsomes (preferentially enriched with endoplasmic reticulum) isolated from bovine pulmonary artery smooth muscle tissue with H2O2 (1 mM) markedly stimulated matrix metalloproteinase activity and also inhibited Na+ dependent Ca2+ uptake. Electron micrograph revealed that H2O2 (1 mM) does not cause any damage to the microsomes. MMP-2...

Treatment of bovine pulmonary artery smooth muscle microsomes with tert-butylhydroperoxide (t-buOOH) (300 microM) markedly stimulated matrix metalloproteinase-2 (MMP-2) activity and enhanced Ca(2+)-ATPase activity and ATP-dependent Ca2+ uptake. Pre-treatment with vit. E (1 mM) and tissue inhibitor of metalloproteinase-2 (TIMP-2) (50 microg/ml) prev...

We have previously indicated that bovine pulmonary artery smooth muscle plasma membrane possesses a complex of 72-kDa gelatinase and TIMP-2 (MMP-2/TIMP-2 complex) [Mol. Cell. Biochem. 258 (2004) 73]. In this paper, we described isolation of MMP-2 from the MMP-2/TIMP-2 complex, characterizations of the isolated MMP-2 and also the complex. MMP-2/TIMP...

Treatment of microsomes (preferably enriched with endoplasmic reticulum) isolated from bovine pulmonary artery smooth muscle tissue with the O2*- -generating system (hypoxanthine (HPX) plus xanthine oxidase (XO)), markedly stimulated matrix metalloproteinase-2 (MMP-2) activity and also enhanced Ca2+ ATPase activity and ATP-dependent Ca2+ uptake. Pr...

Bovine pulmonary artery smooth muscle tissue possesses matrix metalloproteinase-2 (72 kDa gelatinase: MMP-2; E.C. 3.4.24.24) as revealed by immunoblot studies of its plasma membrane suspension with polyclonal MMP-2 antibody. In this report, we described the purification and partial characterization of MMP-2 in the plasma membrane fraction of the sm...

Treatment of bovine pulmonary artery smooth muscle microsomes with peroxynitrite (ONOO-) (100 microM) markedly stimulated matrix metalloproteinase-2 (MMP-2) activity and also enhanced Ca2+ATPase activity and ATP-dependent Ca2+ uptake. Pretreatment of the microsomes with vitamin E (1 mM) and TIMP-2 (50 microg/ml) preserved the increase in MMP-2 acti...

Bovine pulmonary artery smooth muscle tissue possesses the tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) as revealed by immunoblot studies of the cytosolic fraction with polyclonal TIMP-1 antibody. In this report, we described the purification and partial characterization of the inhibitor from the cytosolic fraction of the smooth muscle....

Bovine pulmonary artery smooth muscle possesses the tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) as revealed by Western immunoblot study of its cytosol fraction with bovine polyclonal TIMP-2 antibody. This potent polypeptide inhibitor of matrix metalloproteinases (MMPs) was purified to homogeneity from cytosol fraction of bovine pulmonar...

The matrix metalloproteinases (MMPs) are zinc dependent endopeptidases known for their ability to cleave one or several extracellular matrix (ECM) constituents, as well as non-matrix proteins. They comprise a large family of proteinases that share common structural and functional elements and are products of different genes. All members of this fam...

Matrix metalloproteinases (MMPs) are a major group of enzymes that regulate cell-matrix composition. MMP genes show a highly conserved modular structure. Ample evidence exists on the role of MMPs in normal and pathological processes, including embryogenesis, wound healing, inflammation, arthritis, cardiovascular diseases, pulmonary diseases and can...

Generation of reactive oxygen species (ROS) is a normal process in the life of aerobic organisms. Under physiological conditions, these deleterious species are mostly removed by the cellular antioxidant systems, which include antioxidant vitamins, protein and non-protein thiols, and antioxidant enzymes. Since the antioxidant reserve capacity in mos...

Matrix metalloproteinases (MMPs) are a family of neutral proteinases that are important for normal development, wound healing, and a wide variety of pathological processes, including the spread of metastatic cancer cells, arthritic destruction of joints, atherosclerosis, pulmonary fibrosis, emphysema and neuroinflammation. In the central nervous sy...

Exposure of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant H2O2 (1 mM) stimulated Ca2+ATPase activity. We sought to determine the role of matrix metalloprotease-2 (MMP-2) in stimulating Ca2+ATPase activity by H2O2 in the smooth muscle plasma membrane. The smooth muscle membrane possesses a Ca2+-dependent protease...

A considerable number of experimental, epidemiological and clinical studies are now available which point to an important role of Mg2+ in the etiology of cardiovascular pathology. In human subjects, hypomagnesemia is often associated with an imbalance of electrolytes such as Na+, K+ and Ca2+. Abnormal dietary deficiency of Mg2+ as well as abnormali...

Treatment of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant tert-butylhydroperoxide (t-buOOH) increases Ca2+ATPase activity. The smooth muscle plasma membrane possesses a Ca2+ dependent protease activity in the gelatin containing zymogram having an apparent molecular mass of 72 kDa. The 72 kDa protease activity wa...

We have determined effect of the oxidant peroxynitrite (ONOO-) on Ca2+-dependent matrix metalloprotease-2 (MMP-2) activity and the role of the protease on Ca2+ ATPase activity in bovine pulmonary vascular smooth muscle plasma membrane under ONOO- -triggered conditions. The smooth muscle plasma membrane possesses a 72-kDa protease activity in a gela...

Oxidative stress causes cellular injuries that are mediated, at least in part, by an increase in cytosolic Ca²⁺ concentration [Ca²⁺]i. Disturbances in a variety of mechanisms that normally maintain intracellular Ca²⁺ homeostasis occur during oxidant stress. For example, oxidants, such as hypochlorous acid (HOCL), cause an increase in [Ca²⁺]i that c...

Increasing evidence demonstrated that atherosclerosis is an immunologically mediated disease. Myocardial ischemia/reperfusion injury is accompanied by an inflammatory response contributing to reversible and irreversible changes in tissue viability and organ function. Three major components are recognized as the major contributing factors in reperfu...