Hamlyn, J.M., Blaustein, M.P., Bova, S., DuCharme, D.W., Harris, D.W., Mandel, F., Mathews, W.R. and Ludens, J.H. (1991) Identification and Characterization of a Ouabain-like Compound From Human Plasma. Proc. Natl. Acad. Sci. (USA). 88:6259-6263.

Gottlieb, S.S., Rogowski, A.C., Weinberg, M., Krichten, C.M., Hamilton, B.P. and Hamlyn, J.M. (1992) Elevated concentrations of endogenous ouabain in patients with congestive heart failure. Circulation. 86:420-425.

Manunta, P., Rogowski, A.C., Hamilton, B.P., Hamlyn, J.M. (1992) Ouabain-induced hypertension in the rat: relationships among circulating and tissue ouabain and blood pressure. J. Hypertension. 12:549-560.

Rossi, G.P., Manunta, P., Hamlyn, J.M., Pavan, E., De Toni, R., Semplicini, A., Pessina, A.C. (1995) Endogenous ouabain in primary aldosteronism and essential hypertension: relationship with plasma renin, aldosterone and blood pressure levels. J. Hypertension.13:1181-1191.

Laredo, J., Shah, J.R., Lu, Z-R., Hamilton, B.P., Hamlyn, J.M. (1997) Angiotensin II stimulates secretion of endogenous ouabain from bovine adrenocortical cells via angiotensin type-2 (AT2) ptors. Hypertension. 29 (2):401-407.34.

Shah, J.R., Laredo, J., Hamilton, B.P., Hamlyn, J.M. (1999) Effects of Angiotensin II on Sodium Potassium Pumps, Endogenous Ouabain and Aldosterone in Bovine Zona Glomerulosa Cells. Hypertension. 33 (pt2):373-377.

Manunta, P., Stella, P., Rivera, R., Ciurlino, D., Cusi, D., Ferrandi, M., Hamlyn, J.M., Bianchi, G. (1999) Increased Left Ventricular Mass, Stroke Volume and Ouabain-like Factor in Essential Hypertension. Hypertension. 34:450-456.

Arnon, A., Hamlyn, J.M., Blaustein, M.P. (2000) Sodium entry via Store Operated Channels Modulates Ca2+ Signalling in Arterial Myocytes. Am J Physiol Cell Physiol. 278(1):C163-C173.

Templeton, J.T., Hamlyn, J.M., Hamilton, B.P., Ayotte, J., Majgier-Baranowska, H., Lester, A., Perrealt, H., Marat, K. (2000) Regioselective derivatization of ouabain with trialkylsilyl reagents and selective oxidation of the unprotected alcohols. Steroids. 65:379-386.

Arnon, A., Hamlyn, J.M., Blaustein, M.P. (2000) Ouabain Augments Ca2+ transients in Arterial Smooth Muscle Without Raising Cellular Sodium. Am. J. Physiol. 279:H679-691.

Manunta, P., Hamilton, J., Rogowski, A.C., Hamilton, B.P., Hamlyn, J.M. (2000) Chronic Hypertension Induced by Ouabain but not Digoxin in the Rat: Antihypertensive Effect of Digoxin and Digitoxin. Hypertension Research. 23:S77-85.

Kimura, K., Manunta, P., Hamilton, B.P., Hamlyn, J.M. (2000) Different Effects of In Vivo Ouabain and Digoxin on Renal Artery Function and Blood Pressure in the Rat. Hypertension Arch. 23:S67-76.

Manunta, P., Hamilton, B.P., Hamlyn, J.M. (2001) Structure-activity relationships for the hypertensinogenic activity of ouabain: role of the sugar and lactone ring. Hypertension. 37(2):472-477.

Kawamura, A., Abrell, L., Maggiali, F., Berova, N., Nakanishi, K., Labutti, J., Magil, S., Haupert, G.T., Jr., Hamlyn, J.M. (2001)  Conformational Flexibility of Ouabain and its Biological Implication: Observations with Ouabain 1,5,19- and 1,11,19- Phosphates.Biochemistry. 40(19): 5835-5844.

Manunta, P., Messaggio, E., Ballabeni, C., Sciarrone, M.T., Lanzani, C., Ferrandi, M., Hamlyn, J.M., Cusi, D., Galletti, F., Bianchi, G. (2001)  Plasma Ouabain-Like Factor During Acute and Chronic Changes in Sodium Balance in Essential Hypertensive Patients.Hypertension. 38:198-203.

Aileru, A.A., deAlbuquerque, A., Hamlyn, J.M., Manunta, P., Shah, J.R., Hamilton, M.J., Weinreich, D. (2001) Altered Use-Dependent Synaptic Plasticity in the Sympathetic Ganglia from Acquired and Inherited Forms of Ouabain-Dependent Hypertension. American Journal of Physiology. 281(2):R635-44.

Ward, S.C., Hamilton, B.P., Hamlyn, J.M. (2002) Novel receptors for ouabain in bovine adrenocortical cells and membranes.Hypertension. 39(2):536-542.

Hamlyn, J.M., Laredo, J., Shah, J.R., Lu, Z-R., Hamilton, B.P. (2003) 11-Hydroxylation in the Biosynthesis of Endogenous Ouabain: Multiple Implications. Annals of the New York of Sciences Academy. 986: 685-693.

Wang, J-G., Staessen, J.A., Messaggio, E., Nawrot, T., Fagard, R., Hamlyn, J.M., Bianchi, G., Manunta, P. (2003) Salt, Endogenous Ouabain and Blood Pressure Interactions in the General Population. Journal of Hypertension. 21(8):1475-1481.

Blaustein, M.P., Robinson, S., Gottlieb, S.S., Balke, C.W., Hamlyn, J.M. (2003) Sex, Digitalis, and the Sodium Pump.Molecular Interventions. 3:68-72.

Lighthall, G., Hamilton, B.P., Hamlyn, J.M. (2004) Identification of salt-sensitive genes in the kidneys of Dahl Rats.Journal of Hypertension. 22(8):1487-94.

Lanzani, C., Citterio, L., Jankaricova, M., Sciarrone, M.T., Barlassina, C., Fattori, S., Messaggio, E., Serio, C.D., Zagato, L., Cusi, D.,Hamlyn, J.M., Stella, A., Bianchi, G., Manunta, P. (2005) Role of the adducin family genes in human essential hypertension. Journal of Hypertension. 23(3):543-9.

Manunta, P., Iacoviello, M., Forleo, C., Messaggio, E., Hamlyn, J.M., Lucarelli, K., Bianchi, G., Rizzon, P. and Pitzalis, M.V. (2005) High Circulating Levels of Endogenous Ouabain in the Offspring of Hypertensive and Normotensive Individuals. Journal of Hypertension. 23(9):1677-81.

Zhang, J., Lee, M-Y., Cavalli, M., Chen, L., Berra-Romani, R., Balke, C.W., Bianchi, G., Ferrari, P., Hamlyn, J.M., Iwamoto, T., Lingrel, J.B., Matteson, D.R., Wier, W.G. and Blaustein, M.P. (2005) Sodium pump a2 subunits control myogenic tone and blood pressure in mice. J. Physiol. 569:243-56. PMCID: PMC1464198

Pitzalis, M.V., Hamlyn, J.M., Messaggio, E., Iacoviello, M., Bianchi, G., Rizzon, P., and Manunta, P. (2006) Independent and incremental prognostic value of endogenous ouabain in idiopathic dilated cardiomyopathy. Eur J Heart Fail. 8(2):179-86.

Manunta, P., Hamilton, B.P. and Hamlyn, J.M. (2006) Salt intake and depletion increase circulating levels of endogenous ouabain in normal men. Am J Physiol Regul Integr Comp Physiol. 290(3):R553-9.

Blaustein, M.P., Hamlyn, J.M. and Pallone, T.L. (2007) Sodium pumps: ouabain, ion transport, and signaling in hypertension. Am J Physiol Renal Physiol. 293(1):F438; author reply F439.

Stella P, Manunta P, Mallamaci F, Melandri M, Spotti D, Tripepi G, Hamlyn JM, Malatino LS, Bianchi G, Zoccali C. (2008)  Endogenous ouabain and cardiomyopathy in dialysis patients. J Intern Med. Mar;263(3):274-80. PMCID: PMC3518455

Manunta P, Maillard M, Tantardini C, Simonini M, Lanzani C, Citterio L, Stella P, Casamassima N, Burnier M, Hamlyn JM, Bianchi G. (2008) Relationships among endogenous ouabain, alpha-adducin polymorphisms and renal sodium handling in primary hypertension. J Hypertens. 26(5):914-20.

Cao C, Payne K, Lee-Kwon W, Zhang Z, Lim SW, Hamlyn JM, Blaustein MP, Kwon HM, Pallone TL. (2009) Chronic ouabain treatment induces vasa recta endothelial dysfunction in the rat. Am J Physiol Renal Physiol. 296(1):F98-F106. PMCID: PMC2636913

Manunta P, Ferrandi M, Bianchi G, Hamlyn JM. (2009) Endogenous ouabain in cardiovascular function and disease. J Hypertens.27(1):9-18. PMCID: PMC3518455

Blaustein MP, Zhang J, Chen L, Song H, Raina H, Kinsey SP, Izuka M, Iwamoto T, Kotlikoff MI, Lingrel JB, Philipson KD, Wier WG,Hamlyn JM. (2009) The pump, the exchanger, and endogenous ouabain: signaling mechanisms that link salt retention to hypertension. Hypertension. 53(2):291-8. Epub 2008 Dec 22. Review. No abstract available. PMCID: PMC2727927

Tripodi G, Citterio L, Kouznetsova T, Lanzani C, Florio M, Modica R, Messaggio E, Hamlyn JM, Zagato L, Bianchi G, Staessen JA, Manunta P. (2009) Steroid biosynthesis and renal excretion in human essential hypertension: association with blood pressure and endogenous ouabain. Am J Hypertens. 22(4):357-63. PMCID: PMC3518306

Zhang J, Hamlyn JM, Karashima E, Raina H, Mauban JR, Izuka M, Berra-Romani R, Zulian A, Wier WG, Blaustein MP. (2009) Low-dose ouabain constricts small arteries from ouabain-hypertensive rats: implications for sustained elevation of vascular resistance. Am J Physiol Heart Circ Physiol. 297(3):H1140-50. PMCID: PMC2755988

Pulina MV, Zulian A, Berra-Romani R, Beskina O, Mazzocco-Spezzia A, Baryshnikov SG, Papparella I, Hamlyn JM, Blaustein MP, Golovina VA. (2010) Upregulation of Na+ and Ca2+ transporters in arterial smooth muscle from ouabain-induced hypertensive rats.  Am J Physiol Heart Circ Physiol. 298(1):H263-74. PMCID: PMC2806143

Blaustein MP, Hamlyn JM. (2010) Signaling mechanisms that link salt retention to hypertension: Endogenous ouabain, the Na(+) pump, the Na(+)/Ca(2+) exchanger and TRPC proteins. Biochim Biophys Acta. 2010 Dec;1802(12):1219-29. PMCID: PMC2909369

Manunta P, Messaggio E, Casamassima N, Gatti G, Carpini SD, Zagato L, Hamlyn JM. (2010) Endogenous ouabain in renal Na(+) handling and related diseases. Biochim Biophys Acta. 2010 Dec;1802(12):1214-8. PMCID: PMC3517663

Bignami E, Casamassima N, Frati E, Lanzani C, Corno L, Alfieri O, Gottlieb S, Simonini M, Shah KB, Mizzi A, Messaggio E, Zangrillo A, Ferrandi M, Ferrari P, Bianchi G, Hamlyn JM, Manunta P.  (2013) Preoperative endogenous ouabain predicts acute kidney injury in cardiac surgery patients. Crit Care Med. Mar;41(3):744-55. PMCID: PMC3763989

Song H, Karashima E, Hamlyn JM, Blaustein MP. (2014) Ouabain-digoxin antagonism in rat arteries and neurones. J Physiol. Mar 1;592(Pt 5):941-69. Epub 2013 Dec 16. PMCID: PMC3948557

Simonini M, Lanzani C, Bignami E, Casamassima N, Frati E, Meroni R, Messaggio E, Alfieri O, Hamlyn JM, Body SC, Collard CD; the CABG Genomics Investigators, Zangrillo A, Manunta P.  (2014)  A new clinical multivariable model that predicts postoperative acute kidney injury: impact of endogenous ouabain. Nephrol Dial Transplant. 2014 Jun 11. 2014 Sep;29(9):1696-701. PMCID: PMC4200038

Hamlyn JM, Linde CI, Gao J, Huang BS, Golovina VA, Blaustein MP, Leenen FH. Neuroendocrine humoral and vascular components in the pressor pathway for brain angiotensin II: a new axis in long term blood pressure control. PLoS One. 2014 Oct 2;9(9):e108916. doi: 10.1371/journal.pone.0108916. eCollection 2014. PMCID: PMC4183521.  **NOTE**  This paper was the basis for a joint press announcement from UMB and Univ of Ottawa. In that communication we announced the discovery of a new hormone axis linking the brain with the function of peripheral arteries.

Hamlyn JM. Natriuretic hormones, endogenous ouabain, and related sodium transport inhibitors. Front Endocrinol (Lausanne). 2014 Dec 3;5:199. doi: 10.3389/fendo.2014.00199. eCollection 2014. Review. PMCID: PMC4253959.

Hamlyn JM, Manunta P.  (2015) Endogenous cardiotonic steroids in kidney failure: a review and an hypothesis. Adv Chronic Kidney Dis. 2015 May;22(3):232-44. PMCID:  PMC4410676.

Gatti G, Lanzani C, Citterio L, Messaggio E, Carpini SD, Simonini M, Pozzoli S, Casamassima N, Zagato L, Rampoldi L, Hamlyn JM, Manunta P. (2015) 6C.06: Genes involves in blood pressure response to acute and chronic salt modifications: identification of a new pathway.  J Hypertens. 2015 Jun;33 Suppl 1:e80-1. PMID: 26102939

Lanzani C, Gatti G, Citterio L, Messaggio E, Delli Carpini S, Simonini M, Casamassima N, Zagato L, Brioni E, Hamlyn JM, Manunta P. (2016)Lanosterol Synthase Gene Polymorphisms and Changes in Endogenous Ouabain in the Response to Low Sodium Intake.  Hypertension. 2016 Feb;67(2):342-8. PMCID: PMC4713326 

Blaustein MP, Chen L, Hamlyn JM, Leenen FH, Lingrel JB, Wier WG, Zhang J. (2016) Pivotal role of α2 Na⁺ pumps and their high affinity ouabain binding site in cardiovascular health and disease.  J Physiol. 2016 Jun 28.  PMCID: PMC5088234

Hamlyn JM and Blaustein MP. (2016) Endogenous Ouabain: Recent Advances and Controversies. Hypertension, 2016 Sep;68(3):526-32.  PMCID: PMC4982830

Tentori S, Messaggio E, Brioni E, Casamassima N, Simonini M, Zagato L, Hamlyn JM, Manunta P, and Lanzani C. (2016) Endogenous Ouabain and Aldosterone are Co-elevated in the Circulation of Patients with Essential Hypertension. Journal of Hypertension, 2016 Oct;34(10):2074-80.  PMID: 27457665

In contemporary thinking, our research might be termed; "OUABAINOMICS". The latter term is derived from Ouabain - the name given to an unusual steroid found in certain rare plants primarily of African origin. Ouabain was first used as an arrow poison and is a high affinity reversible inhibitor of the sodium pump that, in high doses, leads mto cardiac arrest. It has been used therapeutically for the acute treatment of shock, cancer, atrial arrhythmias and heart failure. Its clinical utility typically was limited by poor oral bioavailability (<5% of the ingested dose) so it was usually given in small amounts parenterally. In some European countries, large doses are still given by mouth to patients with cardiac conditions. The existence of endogenous analogues had been suspected since the 1930s and indeed had been demonstrated by bioassay methods by many groups (e.g., Rein, Kramer, Gonick, Haddy, Pamnani, Jandyhala). However, and despite various attempts at identification, the nature of the materials remained unclear. In 1983, I began to screen small volumes of human plasma for sodium pump inhibitors. This work was later scaled up and ~ 2 tons of plasma was used to develop the various methods that led to the detection of at least four highly biologically active materials; other compounds potentially present were below the detection limit of the ion transport assay used for screening. The most active of these four materials was isolated and, much to everyone’s surprise, was identified as ouabain or a closely related isomer. Because the plasma donors could not have received ouabain therapeutically - the drug had been withdrawn from clinical use in the USA many years before.  Subsequent work showed that most of the circulating material originated from the adrenal cortex with a typically smaller contribution from a source within the brain. Ouabain was thus the first of the endogenous mammalian sodium pump inhibitors to be identified. Other groups (Goto, Valdes, Haupert, Lichtstein, Bagrov, Takahashi and Schoner) have identified or suggested closely related steroids in mammals. While some work implied the possibility of bioactive isomers of ouabain and especially the 11B epimer, it is clear that the major biologically active compound in humans, cows and rodents is ouabain. In adition to ouabain, two chromatographically distinct isomers have since been described. At physiological concentrations (50-800 pM) ouabain modulates intracellular ion concentrations in discrete sub plasma membrane microdomains without detectably changing ion concentrations in the bulk cell cytosol and alters the function of neurons, cardiac and vascular smooth muscle cells. The remaining outstanding issues include the elucidation of the distal steps in the biosynthetic pathway, and the role of previously undetected ouabain isomers in the brain and their role in physiology and disease.

Biosynthesis and Adrenocortical Secretion of Endogenous Ouabain

Endogenous ouabain is actively secreted by the adrenal gland and to some extent by the brain. Nevertheless, the mechanisms of the synthesis and secretion of endogenous ouabain remain largely unexplored. The early portion of the biosynthetic pathway is similar to that for aldosterone and occurs primarily in the zona glomerulosa cell layer of the adrenal gland. The distal events and intermediates in the biosynthesis remain to be determined.  Once the endogenous ouabain has been synthesized, it is actively transported out of the cell by an unknown mechanism.  The combinination of laboratory studies with clinical studies (Collaborator Dr. Bruce Hamilton) in humans has led to several insgihts into the regulation and significance of endogenous ouabain.

Proteomics and the Biosynthesis of Endogenous Ouabain

In working on the biosynthetic pathway for endogenous ouabain, we developed a series of photo affinity probes, some of which specifically label a number of proteins. Most of these proteins have been identifed by mass spectrometry and some appear to be directly associated with specific reactions in the biosynthetic pathway.

Genetics of Hypertension

The Milan Group at the Ospedale San Raffaele led by Paolo Manunta has shown that one third to one half of patients with common high blood pressure have elevated endogenous ouabain in the circulation. In  parallel with the clinical observations, we developed two new strains of rats in which one strain was hypersensitive to endogenous ouabain and in the later generations spontaneously developed high blood pressure. The counterpart strain was resistant to both endogenous and exogenous ouabain and maintained blood pressures that were  lower than normal and, most interstingly, that did not rise with age. By combining the phenotypes of every animal arising from sib mating for ten generations with information from liver biopsies, we hope to locate key genes that underlie the blood pressure phenotypes. Contact us if you are a molecular geneticist with an interest in this area and have the tools to help eluciate the key genes.

Mechanisms of Ouabain-Induced Hypertension: Is the Brain or the Artery More Important?

The prolonged administration of low doses of ouabain in common used  rodent strains typically induces a sustained hypertension due to elevated total peripheral vascular resistance. The mechanism of the elevated resistance, which is beleived to be functional not structural, is encompassed by three hypotheses. The first is that endogenous ouabain raises blood pressure because it first affects sodium pumps and  secondarliy calcium handling mechanisms in the vascular wall (including the Na/Ca exchanger, the Na/K pump, store-operated channels and the sacroplasmic reticulum). Very substantial evidence from transgenic animals and an array of pharmacological studies supports that hypothesis. The second hypotheis is that the interaction of ouabain with sodium pumps activates signaling cascades and these are critical for the ability of ouabain to raise blood pressure. Very substantial evidence also supports this hypothesis. The third hypothesis, advocated by the Leenen group in Ottawa, is that endogenous ouabain can enter the CNS from the circulation and raise blood pressure via mechanisms that involve brain aldosterone and angiotensin II. Activation of this brain pathway increases sympathetic nerve activity which in turn raises the tone of peripheral arteries. An additional element in this third hypothesis is that small amounts of  ouabain are present endogenously in the CNS due to local biosynthesis at discrete sites. Further local biosynthesis of ouabain can be stimulated bybrain  angiotensin II. Increases in brain ouabain per se can regulate sympathetic outflow and thereby impact blood pressure independent of its circulating levels. Accordingly, my laboratory has  become increasingly enamoured with the role of the brain in setting long-term blood pressure. It should be mentioned also that while sodium pumps are often stated as the primary target of ouabain, other high affinity specific binding sites for ouabain exist in the adrenal cortex (Ward et al 2002) and in the brain. We hope to identify and these sites and determine their roles in the long-term blood pressure effects of ouabain.

Where is Ouabain in the Brain?

The notion that ouabain is made in the brain instantly raises a number of questions: Where is it made, how discrete are the biosynthetic sites, how much is made, what are the local concentrations, are there hotspots, and how far do gradients extend, etc? Once that foundational information is in hand, we can begin to ask about the brain-wide changes in ouabain that occur with age, gender, and neurological disorders, etc. Among all of the analytical techiques, mass spectrometry is the critical technology to address these questions.

We use any method that can answer our questions. Chromatography, Hormone Radioimmunoassays, Tracer methods, Steroid Biosynthesis, Radioreceptor Binding, Cell Culture, Ion Transport, Whole animal hemodynamics, PCR, Photoaffinity labeling, Animal Genetics, as well as NMR and Mass Spectrometry.

• John Templeton - University of Manitoba
• Koji Nakanishi - Columbia Univeristy, New York (deceased)
• Paolo Manunta - University of Milan, Ospedale San Raffaele
• Frans Leenen - University of Ottawa
• Davor Pavlovic - University of Birmingham
• Rifaat El-Mallaakh - University of Louisville

Some Alumni

• Stephen Ward, MD/PhD
• Chris Baba, BS, MS
• Mandi Stevens (Summer fellow)
• Robert Rogowski, BS
• Yasmin Khan, MD
• Gerti Tasko, MD (Visiting Fellow)
• Junjie Gao, Ph.D.
• Andy Yong Liu, PhD
• Brandiese Jacobs, BS. PhD
• Varun Tilva (Summer Student)
• Ian Whitaker, BS, MS
• Loren Gorelick, BS, MS
• Marzieh Keshtkarjahromi, MD

Current

• Pooja Vajpaye BS
• Mitchell Kelly BS