Diabetologia 9 Springer-Verlag 1995

Transcription

1 Diabetlgia (15) 38:72-77 Diabetlgia Springer-Verlag 15 Insulin resistance is assciated with high plasma uabain-like immunreactivity cncentratin in NIDDM T. Wasada 1, H. Kurki 1, M. Naruse 2, H. Arii 1, A. Maruyama 1, K. Katsumri 1, S. Sait 1, Y. Watanabe 3, K. Naruse 2, H. Demura 2, Y. Omri 1 1 Diabetes Center, Tky Wmen's Medical Cllege, Tky, Japan 2 Department f Medicine, Institute f Clinical ndcrinlgy, Tky Wmen's Medical Cllege, Tky, Japan a Mitsubishiyuka BCL, Tky, Japan Summary The aim f the present study was t elucidate the pathphysilgic significance f circulating uabain as a link between insulin resistance (IR) and hypertensin (HT) in NIDDM. uglycaemic (4.5 mml/1) hyperinsulinaemic (36-58 pml/1) clamping was perfrmed using an artificial endcrine pancreas. Plasma uabain-like immunreactivity (OLI) was determined by radiimmunassay using a highly specific antibdy t uabain. HT was defined as systlic bld pressure > 14 mmhg and/r diastlic > mmhg r being treated with antihypertensive agents. The values (mean + SM) f glucse infusin rate (GIR) and plasma OLI were cmpared amng the fur grups classified using IR and HT as factrs. Grup I (IR-/HT-, n=15):gir mg. kg -~ min -1, OLI pml/l, which was nt different frm that in eight nrmal cntrl subjects ( mg. kg -1 - rain -1 and pml/l, respectively); Grup II (IR-/HT+, n = 13): mg. kg -1 min -1, pml/1; GruplII (IR+/HT-, n=14) mg. kg -1 min -1, pml/1 (p <.1 vs Grup I and II); Grup IV (IR+/HT+, n = 15) mg. kg -1 min -1, pml/1 (p <.1 vs Grup I and II), respectively. Six f 57 NIDDM patients studied exhibited very high (> 15 pml/l) plasma OLI cncentratins, shwed marked insulin resistance and were all hypertensive. When analysed as a whle, plasma OLI was negatively crrelated with GIR (p <.1), but was nt crrelated with arterial bld pressure. These results demnstrate that plasma cncentratin f OLI is clsely assciated with the severity f IR but nt with bld pressure elevatin. It is, hwever, pssible that in sme fractin f NIDDM patients with insulin resistance, the elevatin f bld pressure may be causally related t circulating OLI. [Diabetlgia (15) 38: 72-77] Key wrds Ouabain-like immunreactivity, insulin resistance, hypertensin, nn-insulin-dependent diabetes. Received: 13 September 14 and in revised frm: December 14 Crrespnding authr." Dr. T. Wasada, Diabetes Center, Tky Wmen's Medical Cllege, 8-1 Kawada-ch, Shinjuku-ku, Tky 162, Japan Abbreviatins: BSA, Bvine serum albumin; NIDDM, nn-insulin-dependent diabetes mellitus; OLI, uabain-like immunreactivity; GIR, glucse infusin rate; BMI, bdy mass index. Part f this study was presented in the 54th Annual Meeting f the American Diabetes Assciatin, June 11-14, 14, New Orleans, USA The mechanism(s) behind hypertensin in nn-insulin-dependent diabetes mellitus (NIDDM) are prly understd, but sme studies have indicated that sdium retentin may play a majr rle [1-4]. Impaired natriuresis and resultant expansin f extracellular fluid vlume have been reprted t be assciated with hyperinsulinaemia r insulin resistance [5, 6]. Furthermre, insulin resistance has been shwn t accmpany the increased sdium and decreased ptassium cntent in erythrcytes [7], and recent evidence als suggests that high Na+-Li + cuntertransprt is assciated with insulin resistance in patients with essential hypertensin [8]. Thus, intracellular sdium accumulatin, which in turn cu-

2 T. Wasada et al.: Plasma OLI and insulin resistance in NIDDM ples with increased cytslic free calcium t h r u g h Na + e x c h a n g e m a y cause c n t r a c t i n f resistant vessels r sensitizatin f vascular s m t h muscle cells t vascnstrictrs [], t h e r e b y leading t h y p e r t e n s i n [1]. In this cntext, Na activity has b e e n r e p r t e d t be lw in a variety f tissues in insulin-resistant states including diabetes [11-14]. T h e r e f r e, the r e d u c e d activity f s d i u m p u m p s (Na 247 m a y be a k e y m e c h a n i s m linking h y p e r t e n s i n and insulin resistance in N I D D M. Recently, a p t e n t inhibitr f Na 247 Pase was islated f r m h u m a n plasma and f u n d t b e indistinguishable f r m u a b a i n [15-17], althugh this nvel e v i d e n c e has b e e n c h a l l e n g e d by r e c e n t studies [18, 1]. In the p r e s e n t study, t h e r e f r e, we m e a s u r e d plasma uabain-like i m m u n r e a c t i v i t y ( O L I ) cnc e n t r a t i n s in patients with N I D D M and severe insulin resistance r n r m a l insulin sensitivity t elucidate w h e t h e r u a b a i n is invlved in the p a t h p h y silgy f h y p e r t e n s i n assciated with insulin resistance. Subjects and methds Subjects. We studied 57 patients with NIDDM and eight healthy cntrl subjects. Nne f these subjects had vert diabetic cmplicatins except early backgrund retinpathy and intermittent prteinuria in sme patients. All subjects were in stable health and they had n symptms f cngestive heart failure r edema f any aetilgy. Since NIDDM patients exhibit great variatin in their individual sensitivity t insulin (GIR: mg. kg-1. min-1) (Fig. l), we randmly selected the subjects in the present study frm bth the highest quartile grup (GIR>5.2 mg. kg-1. min -1) and the lwest quartile grup (GIR < 2.2 mg. kg 1. min 1). As shwn in Table 1, NIDDM patients were divided int fur subgrups accrding t the degree f insulin resistance and hypertensin. Hypertensin was defined as average systlic pressure f greater than 14 mmhg and/r average diastlic pressure f greater than mmhg n at least three ccasins preceding the euglycaemic clamp study r being treated with antihypertensive drugs. The fur subgrups were matched fr bdy mass index (BMI) and fasting bld glucse level. The mean ages (+ SM) f cntrl subjects and Grup I were significantly lwer than thse f ther grups, but there was n difference amng Grups II, III and IV. Glycaemic cntrl (HbAlc) and type f treatment fr diabetes were similar amng the grups. Calcium antagnists were used in 18 f 28 hypertensive patients either alne r in cmbinatin with ther antihypertensive drugs, fllwed by beta-blckers in six patients and angitensin-cnverting enzyme inhibitrs in fur patients. Diuretics were used in nly tw patients. N patient had been given any kind f digitalis treatment. This study was apprved by the Institutinal Cmmittee f the Diabetes Center, TWMC, and infrmed cnsent was btained frm each f the subjects studied. uglycaemic hyperinsulinaemic clamp technique. After an vernight fast and discntinuatin f all the medicatins n the study mrning, euglycaemic hyperinsulinaemic clamping was perfrmed using an artificial endcrine pancreas (Nikkis O (-- 8. c r = -.48 (n = 134) p <.1 7. '7 == ~ % l 5. n- 4. (3 3. ~ 8 % g ~176. 6g~~ ~,; 2. i m 176 : ~ 1. J I t I 25 F a s t i n g p l a s m a IRI ~ U / m l ) Fig.1. Crrelatin between GIR values and fasting plasma IRI cncentratins in 134 NIDDM patients withut insulin therapy. There is a negative crrelatin between these tw parameters (r = -.48, p <.1) and als marked individual variatin in GIR levels (GIR: mg. kg-1 rain-l). indicates besity > 27 kg/m 2 (male), > 25 kg/m2 (female) STG-22, Nikkis C., Tky, Japan). A primed-cntinuus infusin f shrt-acting insulin (Nvlin R, Nv-Nrdisk Pharma C., Cpenhagen, Denmark) at 1.12 mu kg-1 min-1 was started and peripheral venus plasma glucse was clamped at 4.5 mml/1 by infusing varying dses f 1 % glucse accrding t the algrithm riginally described by DeFrnz et al. [2]. A steady-state f euglycaemia was maintained ver 6 min, and the average rate f glucse infusin (GIR, mg. kg 1. min-1) during the final 3 min f the study was used as an index fr insulin resistance in the whle bdy. Steadystate plasma insulin cncentratins were pml/1. Bld samples were drawn fr measurements f insulin (IRI), C-peptide (CPR) and OLI befre and at apprpriate intervals during euglycaemic clamping. Radiimmunassay f plasma OLL Plasma OLI was measured by a recently develped highly specific radiimmunassay [21]. In brief, bld samples cllected in chilled tubes cntaining Na2DTA (4 mml/1) were centrifuged at 4~ and plasma was stred at - 7 ~ until assayed. One ml f plasma sample was applied t a Sep-Pak C18 cartridge (Waters Assciates, Milfrd, Mass., USA) and eluted with 75 % ethanl. The extracts were evaprated, lyphilized, and re-disslved in the assay buffer fr radiimmunassay. The antiserum used in the present assay was raised in New Zealand white rabbits against uabain (Sigma Chemical C., St. Luis, M., USA) cnjugated t bvine serum albumin (BSA). The crss-reactivity f this antibdy was.2 % with digxin, and thse with BSA, rhamnse, hydrcrtisne, and aldsterne were less than.7 %. The sensitivity f the radiimmunassay was.1 pml/tube with 5 % displacement at.24 pml/ tube. In this radiimmunassay, the dilutin curves f plasma extracts were parallel t the standard curve fr authentic uabain. On the reverse-phase HPLC with a linear gradient f acetnitrile/trifluracetic acid, plasma OLI was identified as a single majr peak with the retentin time crrespnding t that f uabain. The recvery rate f uabain added t the plasma sample befre extractin with Sep-Pak was apprximately 5 %. The intra- and interassay cefficients f variatin at a cncentratin f 25 pml/1 were 11.5 and 17.5 %, respectively.

3 74 T. Wasada et al.: Plasma OLI and insulin resistance in NIDDM Table 1. Clinical and metablic characteristics f the grups f study subjects NIDDM patients Grup 1 1I III IV (IR/HT) (-/-) (-/+) (+/-) (+/+) n (male : female) 15 (12:3) 13 (*4) 14 (1:4) Age (years) ~ ab ~b BMI (kg/m 2) ~ Duratin (years) FPG (mml/1) HbAa: (%) r.5.6 SBP (mmhg) f f DBP (mmhg) g g FIRI (~tu/ml) h~ GIR (mg. kg -1 rain -1) j k l Micr/Macr-AU 4/15 3/13 3/14 Treatment Diet alne 6/15 7/13 7/14 OHA 4/15 1/13 4/14 Insulin 5/15 5/13 3/14 Anti-HT 7/13 Values are means + SM. 1R, insulin resistance; HT, hypertensin; BM1, bdy mass index; FPG, fasting plasma glucse; SBP, systlic bld pressure; DBP, diastlic bld pressure; FIRI, fasting plasma insulin; GIR, glucse infusin rate, Micr/Macr-AU, micralbuminuria (3-3 mg/day) r macralbuminuria (> 3 mg/day); OHA, ral hypglycaemic agents; Anti-HT, ant agents; IR-/HT+, fr exam- 15 (1:5) 61.3 ab d hi 1.7 I 5/15 6/15 4/15 5/15 14/15 m Nrmal cntrl subjects 8 (4:4) pie indicates the absence f IR and the presence f HT; ap <.5-.1 vs cntrl; bp <.5-.1 vs 1; :p <.5 vs cntrl; dp <.5 vs 11; ~p <.5 VS 111 and 1V; ~p <.1 vs 11 and 1V; gp<.1 vs I1 and IV; hp<.1 vs cntrl; ip<.5 vs 1I; Jp<.2 vs II; kp<.5 vs cntrl; lp <.1 vs 1, I1 and cntrl, mp<.5 vs 11 Statistical analysis Data were expressed as means SM and statistically analysed by the Mann-Whitney test, the chi-square test, and the nn-linear r linear regressin analysis, as apprpriate. The level f significance was set atp <.5. Results "6 CL m.--j (n 3 8 I * * ' 7 6OO Plasma OLI cncentratins were cmpared amng the grups (Fig. 2). The mean level fr eight nrmal glucse-tlerant subjects was pml/1. The highest level was fund in the Grup IV with bth insulin resistance and hypertensin (73.1_ 17.1 pml/1). The lwest level was in Grup I with neither insulin resistance nr hypertensin (13.8 _+ 2. pml/1), and was nt significantly different frm that in the nrmal cntrl grup. Thse in Grups II and III with either hypertensin r insulin resistance were intermediate between these tw values (172.5 _ 35., _ pml/l, respectively). Very high plasma OLI levels (greater than 15 pml/1) were fund in six patients, all f whm were in the insulin-resistant grups (Grups III and IV). The clinical features f these six patients are presented in Table 2. The mean plasma OLI level was elevated in the hypertensive grups cmpared with the nrmtensive cunterpart, i.e. Grup I vs II, and Grup III vs IV, but the differences were nt statistically significant in either pair. There was an inverse 1 O Nrmal I 1I ~ 1V cntrl NIDDM patients subjects Fig.2. Plasma OLI cncentratins in nrmal cntrl subjects and NIDDM patients. NIDDM patients were divided int fur subgrups by the presence (+) r absence (-) f insulin resistance (IR) and hypertensin (HT): Grup I (IR-/HT-), II (IR-/HT+), III (IR+/HT-), and IV (IR+/HT+). The difference was significant between Grup I and II and Grup III and IV, while thse between nrmal cntrl subjects and Grup I and II, between Grup I and Grup II, and between Grup III and Grup IV were nt significant. *p <.5; **p <.1; ***p <.1 crrelatin between plasma OLI levels and the GIR values when all subjects were cmbined (p <.1, n = 65, Fig. 3), and even when the extremely high values f OLI (> 15 pml/l) were excluded (p <.1, n = 5). Hwever, n crrelatin was fund between plasma OLI and bld pressure.

4 T. Wasada et al.: Plasma OLI and insulin resistance in NIDDM 75 Table 2. Clinical prfile f patients with very high (> 15 pml/1) plasma levels f OLI I. Y.I. N.T. T.W. H.T. K.H. Age (years): 71 :Female 5:Male 65 :Female 64:Female 6:Female 63:Female Sex (male/female) BMI (kg/m 2) HbAlc (%) Hypertensin Drug therapy Ca++-ant. nne Ca++-ant. Ca++-ant. Ca++-ant. nne diuretic (lw salt diet) AC-inh. diuretic (lw salt diet) diuretic GIR (mg. kg -1 rain -1) PRA (ng. ml 1. h-t) PAC (ng/dl) Diabetes therapy SU Diet Insulin SU SU Insulin Past medical histry OMI CHF OMI Hypertensin was defined by systlic bld pressure > 14mmHg and diastlic > mmhg r drug treatment. PRA, Plasma renin activity (nrmal:.5-3. ng. ml t. h-l); PAC, plasma aldsterne cncentratin (nrmal: ng/ dl); SU, sulfnylurea; OMI, ld mycardial infarctin; CHF, cngestive heart failure _1 (/ O 5 25 ~ ~~ ~ m O~ I [ I [ 1 I I I I I I GIR (rag. kg -1. rain -1) Fig.3. Crrelatin between GIR and plasma OLI levels in all subjects studied (eight nrmal cntrl subjects and 57 patients with NIDDM). A nn-linear regressin analysis shwed a significant relatinship between plasma OLI (Y) and GIR (X): Y = X + 7.8X 2, r =.528 (p <.1). When the extremely high values f OLI (> 15 pml/l) were excluded, a linear negative crrelatin was fund between them (r = ~).487, p <.1, n = 5) Discussin The present study demnstrated that plasma cncentratins f OLI are significantly higher in insulin-resistant NIDDM patients than in insulin-sensitive patients r nrmal glucse-tlerant subjects, while there was n difference between the latter tw grups. Furthermre, plasma OLI cncentratin shwed a significant negative crrelatin with the GIR value, when analysed in each subject. T ur knwledge, the relatinship between insulin resistance and plasma levels f OLI has nt been explred directly in patients with NIDDM. Our values f plasma OLI cncentratin were cmparable t thse reprted by ther investigatrs [22, 23]. Hamlyn's grup [22] reprted that the plasma uabain cncentratin ranges frm belw 2 t 7 pml/1 in mst nrmal individuals, but in sme patients t as high as 1 nml/1 r even higher. Similarly, a mre than 5-fld variatin in plasma levels f OLI was fund in the present grup, and in six patients plasma OLI levels exceeded mre than 15 pml/l. These patients shared severe insulin resistance and hypertensin, but therwise there was n clinical marker that characterized this particular subgrup f patients. Since the cncentratins f uabain nrmally present in the circulatin have been demnstrated t be active in mdulating vascular cntractility [24], this may have pathphysilgic implicatins in hypertensin assciated with insulin resistance. Andrnic et al. [25] estimated plasma cncentratins f immunreactive "endgenus digxin-like factr (DLF)", in 14 hypertensive and 12 nrmtensive subjects with besity and glucse intlerance. In their studies, hypertensive subjects shwed higher plasma DLF levels and lwer plasma glucse/insulin rati than nrmal subjects, indicating a pssible assciatin between plasma DLF levels and the degree f insulin resistance. The results f the present study are well in agreement with their findings. Hwever, insulin resistance can be mre reliably evaluated by the euglycaemic hyperinsulinaemic clamp methd, and plasma digitalis-like substance can nw be determined mre directly with specific radiimmunassay fr uabain. Our findings, therefre, prvide direct and better evidence t supprt the clse

5 76 T. Wasada et al.: Plasma OLI and insulin resistance in NIDDM relatinship between plasma OLI and insulin resistance. In ur study, hwever, the relatinship between hypertensin and plasma OLI was nt significant. Plasma OLI tended t be higher in the hypertensive grup, but the difference was nt statistically significant between the grups with cmparable insulin resistance. Therefre, plasma OLI culd nt be cncluded t play a rle in the develpment f hypertensin frm the results f the present study. Hwever, it has since been reprted that plasma OLI levels are lwered after antihypertensive treatment in patients with essential hypertensin [21], plasma levels f OLI in ur patients culd be underestimated, and, thus, may bscure the pssible differences between the grups with and withut hypertensin. Devynck et al. [26] reprted that plasma ptency f DLF measured by its interfering activity with 3H-uabain binding t erythrcyte sdium pumps was significantly crrelated with the rate f urinary sdium utput in patients with essential hypertensin. Therefre, elevated plasma OLI may be part f a cmpensatry mechanism fr impaired natriuresis secndary t insulin resistance. This is in accrdance with the bservatins that salt-lading and expansin f plasma vlume induce a rise in plasma levels f uabain in experimental animals and humans [27], and that elevated levels f uabain are likely t be fund in essential hypertensin characterized by suppressed plasma renin activity [28, 2]. Ouabain pssibly ppses the ability f insulin t stimulate tubular reabsrptin f sdium in the kidney. On the ther hand, in the cardivascular muscle cells, uabain causes an increase in intracellular sdium cncentratin, which in turn reduces the Na+/ Ca ++ cuntertransprt activity and results in increased cytslic free calcium and decreased free magnesium, thus leading t ptentiatin f cardivascular reactivity [, 1, 3]. It has been reprted that Na activity is reduced in a variety f tissues in insulin-resistant states; human essential hypertensin [11, 12], besity [13], and experimental mdels f diabetes [14]. Therefre, ur findings may partly explain the reductin f Na 247 activity accmpanying insulin resistance. Mrever, reduced sdium pump activity has been prpsed t exaggerate neural stimulatin and nrepinephrine verflw [31] which, tgether with increased peripheral vascular resistance, might explain the develpment f hypertensin assciated with insulin resistance. Recently, the parenteral administratin f uabain ver a 4- t 6-week perid was reprted t cause chrnic hypertensin in rats [32]. Hwever, recent studies cast dubts abut the presence f uabain in human plasma and its pathphysilgical rle in the develpment f hypertensin [18, 1]. Therefre, further effrts shuld be made t elucidate whether an endgenus natriuretic and hypertensingenic sub- stance is a true uabain, and whether uabain culd be a link between insulin resistance and hypertensin in NIDDM. References 1. De Chatel R, Weidmann P, Hammer Jet al. (177) Sdium, renin, aldsterne, catechlamines, and bld pressure in diabetes mellitus. Kidney Int 12: O'Hare JA, Ferris JB, Brady D, Twmey B, O'Sullivan DJ (185) xchangeable sdium and renin in hypertensive diabetic patients with and withut nephrpathy. Hypertensin 7 [Suppl II]: Weidmann P, Beretta-Piccli C, Trst BN (185) Pressr factrs and respnsiveness in hypertensin accmpanying diabetes mellitus. Hypertensin 7 [Suppl II]: Feldt-Rasmussen B, Mathiesen R, Deckert T et al (187) Central rle fr sdium in the pathgenesis f bld pressure changes independent f angitensin, aldsterne and catechlamines in type 1 (insulin-dependent) diabetes mellitus. Diabetlgia 3: DeFrnz RF (181) The effect f insulin n renal sdium metablism, a review with clinical implicatins. Diabetlgia 21: Halkin H, Mdan M, Shefi M, Almg S (188) Altered erythrcyte and plasma sdium and ptassium in hypertensin, a facet f hyperinsulinemia. Hypertensin 11: Mdan M, Halkin H, Almg S et al. (185) Hyperinsulinemia, a link between hypertensin, besity and glucse intlerance. J Clin Invest 75: Dria A, Firett P, Avgar Aet al. (11) Insulin resistance is assciated with high sdium-lithium cuntertransprt in essential hypertensin. Am J Physil 261: Swers JF, Khury S, Standley P, Zemel P (11) Mechanisms f hypertensin in diabetes. Am J Hypertens 4: Resnick LM (13) Inic basis f hypertensin, insulin resistance, vascular disease, and related disrders: the mechanism f "syndrme X". Am J Hypertens 6:123 S-134 S 11. Garay RP, Dagher G, Permllet MG, Devynck MA, Meyer P (18) Inherited defect in Na+-K ctransprt system in erythrcytes frm essential hypertensive patients. Nature (Lnd) 284: Hiltn PJ (186) Cellular sdium transprt in essential hypertensin. N ngl J Med 314: De Luise M, Blackman GL, Flier JS (18) Reduced activity f the red-cell sdium-ptassium pump in human besity. N ngl J Med 33: Chen MP, Dasmahapatra A, Shapir (185) Reduced glmerular sdium/ptassium adensine-triphsphatase activity in acute streptzcin diabetes and its preventin by ral srbinil. Diabetes 34: Ludens JH, Clark MA, DuCharme DW et al. (11) Purificatin f an endgenus digitalislike factr frm human plasma fr structural analysis. Hypertensin 17: Mathews WR, DuCharme DW, Hamlyn JM et al. (11) Mass spectral characterizatin f an endgenus digitalislike factr frm human plasma. Hypertensin 17: Hamlyn JM, Blaustein MP, Bva Set al. (11) Identificatin and characterizatin f a uabain-like cmpund frm human plasma. Prc Natl Acad Sci USA 88: Rse AM, Valdes R Jr (14) Understanding the sdium pump and its relevance t disease. Clin Chem 4:

6 T.Wasada et al.: Plasma OLI and insulin resistance in NIDDM Dris PA, Jenkins LA, Stcc DM (14) Is uabain an authentic endgenus mammalian substance derived frm the adrenal? Hypertensin 23: DeFrnz RA, Tbin JD, Andres R (17) Glucse clamp technique; a methd fr quantifying insulin secretin and resistance. Am J Physi1237: Naruse K, Naruse M, Tanabe Aet al. (14) Des plasma immunreactive uabain riginate frm the adrenal gland? Hypertensin 23 [Suppl I]: Hamlyn JM, Manunta P (12) Ouabain, digitalis-like factrs and hypertensin. J Hypertens 1 [Suppl 7]: S -S Manunta P, Hamiltn BS, Pruce, Hamlyn JM (12) High dietary sdium raises plasma levels f uabain in nrmal man. J Hypertens 1 [Suppl 6]: 6 (Abstract) 24. Blaustein MP (13) Physilgical effects f endgenus uabain: cntrl f intracellular Ca ++ stres and cell respnsiveness. Am J Physi1264: C 1367-C Andrnic G, Mule G, Mangan MT et al. (12) Insulin resistance and endgenus digxin-like factr in bese hypertensive patients with glucse intlerance. Acta Diabetl 28: Devynck MA, Pernllet MG, Meyer P (187) ndgenus digitalis-like cmpunds in essential and experimental hypertensin. Int J Rad Appl Instrum (B) 14: Gruber KA, Whitaker JM, Buckalew VM Jr (18) ndgenus digitalis-like substance in plasma f vlume-expanded dgs. Nature 287: Haddy FJ, Pamnani MB (185) vidence fr a circulating endgenus Na pump inhibitr in lw renin hypertensin. Fed Prc 44: Hamlyn JM, Levinsn PD, Ringel R et al. (185) Relatinships amng endgenus digitalis-like factrs in essential hypertensin. Fed Prc 44: Blaustein MP, Ashida T, Gldman WF, Wier WG, Hamlyn JM (186) Sdium/calcium exchange in vascular smth muscle; a link between sdium metablism and hypertensin. Ann NY Acad Sci 488: Tsuda K, Tsuda S, Shima H, Masuyama Y (18) Facilitatry effects f uabain and digitalis-like substance n adrenergic transmissin in hypertensin. Am J Hypertens 2: Yuan C, Manunta P, Chen S, Hamlyn JM, Pamnani MB (12) Hypertensin induced by chrnic uabain administratin in rats (Abstract 48) FASB J 6:A45