John M. Hamlyn
685 West Baltimore St.
HSF I 580A
Short Bio: Made in Britain. Favorites: tea, sushi, good Italian and Indian food, motorcycles, wine, music, guitars, Caribbean, friendly folks, etc. Collaborates freely.
Real Bio: I received my Ph.D. in Biochemistry in 1979, from what is now called the Glasgow Caledonia University. From 1979-1981, I was a postdoctoral fellow in Alan Senior's laboratory in the Department of Biochemistry at the University of Rochester, NY. There, I worked on the biochemistry of pancreatic bicarbonate secretion. I decided to switch from biochemistry to physiology because I realized I was interested in high blood pressure and so I came to the Department of Physiology at the University of Maryland to work on that problem. My work led to the discovery of endogenous ouabain. In the years that followed, I was fortunate to be involved with work going from molecules all the way through to clinical studies.
Currently, I am a Professor, a full member of the Graduate School, and a faculty member of the NIH training program in Integrative Membrane Biology.
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. It has been used therapeutically for the acute treatment of shock, cancer, atrial arrhythmias and heart failure. Its clinical utility was limited by poor oral bioavailability (<5% of the ingested dose) so it was typically 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 inhibitors of the sodium pump 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 inhibitors of the sodium pump. This work was later scaled up so that ~ 2 tons of plasma was eventually used and found four highly biologically active materials were detected. The most active of these materials was isolated and much to everyone’s surprise was identified as ouabain or a closely related isomer. Because the blood donors could not have received ouabain therapeutically - the drug had been withdrawn from clinical use in the USA many years before - the presence of such a compound in the human circulation was most mysterious. Subsequent work showed that it originated from the adrenal cortex. 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. At low, physiologically relevant concentrations, like ouabain, the isolated human analog (endogenous "ouabain") affects intracellular ion concentrations in discrete sub plasma membrane microdomains without changing ion concentrations in the bulk cell cytosol. Endogenous ouabain alters the function of neurons, cardiac and vascular smooth muscle cells. At the moment, our goals are to determine the biosynthetic pathway, show how secretion is regulated, and determine the molecular events by which it raises blood pressure (see below for details). We are also interested in the possibility of previously undetected ouabain isomers and their role in physiology and disease.
Biosynthesis and Adrenocortical Secretion of Endogenous Ouabain
Endogenous ouabain is actively secreted by the adrenal gland. But the way in which the synthesis and secretion of endogenous ouabain occurs and is controlled is not clear. Our work shows that the early portion of the biosynthetic pathway shares some features with the pathway for aldosterone and occurs primarily in the zona glomerulosa cell layer. The distal events and intermediates in the biosynthesis of endogenous ouabain are unknown. Accordingly, we use fresh adrenal glands and cell culture systems to explore the biosynthetic pathway. Once the endogenous ouabain has been synthesized, it is transported out of the cell. We combine laboratory studies with clinical studies (Collaborator Dr. Bruce Hamilton) in humans that look at how the secretion of endogenous ouabain is regulated. While the impression from the cell studies and the clinical data are often in harmony, there are several phenomena specific to the cultured cells that do not appear to be physiologically significant in humans - hence the reliance on a combined approach with cell biology and clinical investigation.
Proteomics and the Biosynthesis of Endogenous Ouabain
In working on the biosynthetic pathway for endogenous ouabain we have begun to probe for some of the enzymes involved using expression array technology. If this approach proves to be useful we expect to isolate the enzymes for the various transformations, study their structures and catalytic mechanisms and the way in which they are regulated.
Genetics of Hypertension
We have proposed that a primary event in the development of common high blood pressure in at least one third to one half of patients with essential hypertension involves the elevation of endogenous "ouabain" in the circulation. This view is based upon a series of clinical studies involving the Milan Group at the Ospedale San Raffaele now led by Paolo Manunta. To follow up on some of the implications of the clinical observations, we developed two new strains of rats. One of the strains is hypersensitive to endogenous ouabain and spontaneously develops high blood pressure. The counterpart strain is resistant to both endogenous and exogenous ouabain and maintains blood pressures that are lower than normal and that do not rise with age. We have studied the phenotypes of every animal arising from sib mating for ten generations. We hope to utilize high throughput genomic technologies to locate the key genes for the blood pressure phenotypes. Those genes should inform on function, may have utility as disease markers, and might be useful therapeutic targets.
Mechanisms of Ouabain Induced Hypertension
How do endogenous ouabain and plant ouabain actually raise blood pressure? Hemodynamic studies show that total peripheral vascular resistance is raised in the hypertensive rats. But by what mechanism is the vascular resistance elevated? There are two prevailing hypotheses which are not mutually exclusive. The first hypothesis proposed by Blaustein in Baltimore is that endogenous ouabain raises blood pressure because it can directly affect calcium handling mechanisms in the vascular wall (including the Na/Ca exchanger, the Na/K pump and store operated channels). There is now very substantial evidence from transgenic animals and an array of pharmacological studies supporting that hypothesis. In addition to inhibition of the sodium pump, work from Askari and Xie shows also that the interaction of ouabain with sodium pumps activates signaling cascades. The roles of pump inhibition vs signaling in the acute versus chronic blood pressure responses to ouabain are not yet known. The second hypothesis, advocated by the Leenen group in Ottawa, is that endogenous ouabain raises blood pressure by tonically affecting the function of sodium pumps in certain groups of neurons in the hypothalamus which, in turn, allow increased amounts of sympathetic nerve activity to reach the peripheral vasculature. One interesting idea is that very small amounts of endogenous ouabain may be synthesized within the CNS itself (although not proven) and might regulate sympathetic outflow and blood pressure independent of the blood levels. In addition to sodium pumps there may be new kinds of receptors for ouabain. Along those lines, we described novel high affinity binding sites for ouabain that were highly enriched in the adrenal cortex (Ward et al 2002) and we have found similar sites in the brain. The identity and function of these sites is not yet clear.
Lab Techniques and Equipment:
Basically 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.
Some of our key extramural collaborators:
- Stephen Ward, MD/PhD (PhD completed 2002)
- Chris Baba, MS (Senior Technician)
- Mandi Stevens (Summer fellow)
- Robert Rogowski, BS (Research Specialist)
- Yasmin Khan, MD (Endocrinology Fellow)
- Gerti Tasko, MD (Visiting Fellow)
- Pratik Dash (Summer student)
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.
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
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.
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.
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
. 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
. 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
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
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
Arnon, A., Hamlyn, J.M.,
Blaustein, M.P. (2000) Ouabain Augments Ca2+ transients in Arterial Smooth Muscle Without Raising Cellular Sodium
. Am. J. Physiol.
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.
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.
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
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
. 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
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
Ward, S.C., Hamilton, B.P., Hamlyn, J.M
. (2002) Novel receptors for ouabain in bovine adrenocortical cells and membranes
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
Blaustein, M.P., Robinson, S., Gottlieb, S.S., Balke, C.W., Hamlyn, J.M.
(2003) Sex, Digitalis, and the Sodium Pump
Lighthall, G., Hamilton, B.P., Hamlyn, J.M.
(2004) Identification of salt-sensitive genes in the kidneys of Dahl Rats
.Journal of Hypertension
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
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
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
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
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.
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
, 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.
. 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.
, Manunta P. Endogenous cardiotonic steroids in kidney failure: a review and an hypothesis
. Adv Chronic Kidney Dis. 2015 May;22(3):232-44. doi: 10.1053/j.ackd.2014.12.005. Review. 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. 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. doi: 10.1097/01.hjh.0000467570.18584.13. PubMed PMID: 26102939