Peter A. Doris

Circulating bufodienolide and cardenolide sodium pump inhibitors in preeclampsia

OBJECTIVEnTo determine plasma levels of the endogenous bufodienolide Na+/K+ ATPase inhibitor, marinobufagenin-like factor (MBG), in normotensive pregnancy and in preeclampsia, to compare changes of MBG with that of ouabain-like compound (OLC), and to characterize the purified MBG immunoreactive factor from preeclamptic plasma.nnnDESIGN AND METHODSnConsecutive sample study. The levels of MBG and OLC compounds were measured in extracted plasma by solid phase fluoroimmunoassays. MBG and ouabain immunoreactive materials were partially purified from preeclamptic plasma via reverse-phase high-performance liquid chromatography (HPLC) and studied for their ability to cross react with MBG and ouabain antibodies, and to inhibit the Na+/K+ ATPase from human mesenteric arteries. Vasoconstrictor effect of authentic MBG was studied in isolated rings of human umbilical arteries.nnnRESULTSnIn 11 nonpregnant control individuals, plasma concentrations of MBG and OLC were 0.190+/-0.04 nmol/l and 0.297+/-0.037 nmol/l, respectively. In the third trimester of noncomplicated pregnancy (n = 6), plasma MBG increased (0.625+/-0.067 nmol/l, P<0.05), and OLC did not (0.32+/-0.07 nmol/l). In 15 patients with preeclampsia, plasma levels of both MBG and OLC increased dramatically (2.63+/-0.10 nmol/l and 0.697+/-0.16 nmol/l, respectively, P<0.01 versus both control groups). When fractionated by reverse phase HPLC, OLC was eluted by 18% acetonitrile, and MBG by 48% acetonitrile. Serially diluted samples of MBG and OLC immunoreactive materials from HPLC fractions reacted with MBG and ouabain antibody in solid phase immunoassay in a concentration dependent fashion. Authentic MBG caused contractile responses of isolated rings of human mesenteric arteries in a concentration-dependent manner. Similarly to the authentic MBG, HPLC purified MBG immunoreactive material from preeclamptic plasma inhibited Na+/K+ ATPase purified from human mesenteric artery.nnnCONCLUSIONSnOur observations demonstrate the coexistence of two endogenous cardiotonic steroids in preeclamptic plasma, a more polar OLC and a less polar MBG-like compound. Substantial increases in plasma OLC and MBG immunoreactivity in preeclampsia, along with the vasoconstrictor properties of authentic MBG and Na+,K+ ATPase inhibitory activity of human MBG immunoreactive factor, suggest, that in preeclampsia, plasma concentrations of MBG are enough to substantially inhibit the sodium pump in cardiovascular tissues, and are in accordance with the views attributing endogenous digitalis-like factors a pathogenic role in the preeclamptic hypertension.

Ouabain- and Marinobufagenin-Induced Proliferation of Human Umbilical Vein Smooth Muscle Cells and a Rat Vascular Smooth Muscle Cell Line, A7r5

Background—We studied the growth-promoting effects of 2 sodium pump–selective cardiotonic steroids, ouabain and marinobufagenin, on cultured cells from vascular smooth muscle (VSMCs) from human umbilical vein and a rat VSMC line, A7r5. Methods and Results—Both ouabain and marinobufagenin activated proliferation of these cells in a concentration-dependent manner, reflecting the cardiotonic steroid sensitivity of the specific &agr;1 subunit contained within each cell source. The observed effective concentration ranges of both compounds was below that necessary to induce cytoplasmic ion alterations by sodium pump inhibition. Conclusions—These data indicate that the ouabain-activated proliferative effect previously observed in canine VSMCs occurs in other VSMC sources. This growth effect seems to be initiated by drug interaction with the sodium pump, reflected by the affinity of the steroid for the pump, and is independent of altered transmembrane ionic gradients.

Endogenous Marinobufagenin-Like Factor In Acute Plasma Volume Expansion

The aim of the study was to further investigate the nature of endogenous digitalis-like factors stimulated by acute plasma volume expansion (VE). 10 male Fisher 344XNB rats were given intravenous (iv) saline infusion (4% of the body mass) for 30 min, which caused a fall in % hematocrit (35.9+/-0.69 vs. 41.8+/-1.05; vs. 10 controls, P<0.01). EDLF was measured in C-18 reverse-phase extracted (32%+80% acetonitrile) tissues. VE was associated with an increase in plasma marinobufagenin-like factor (MLF)(0.49+/-0.05 vs. 0.2+/-0.06 nmol/L, P<0.01) and pituitary ouabain-like compound (OLC) (30.9+/-3.12 vs. 3.2+/-2.3 pmol/g, P<0.01). Plasma OLC decreased (0.087+/-0.018 vs. 0.21+/-0.04 nmol/L, P<0.02), and pituitary MLF did not change (0.05+/-0.01 vs. 0.07+/-0.02 pmol/g) after VE. Chloroform extracted urine from 5 volume-expanded male mongrel dogs was fractionated on reverse-phase HPLC columns in a linear gradient of 0-80% acetonitrile. The material cross-reacting with marinobufagenin antibody eluted from HPLC column as a single peak, demonstrated absorbance at 300 nm, and was distinct from ouabain-like material. Digoxin antibodies interacted with both marinobufagenin-like and ouabain-like immunoreactive material. These results provide further evidence for the presence of a bufodienolide EDLF in the mammals, and demonstrate that EDLF response to VE includes stimulation of brain OLC and plasma bufodienolide.

Vasopressin and central integrative processes

Anatomical studies have suggested that the posterior pituitary peptide, vasopressin (VP, antidiuretic hormone) may be released at central neural sites other than the neurohypophysis. Immunohistochemical techniques allow VP to be visualized at numerous sites in the central nervous system (CNS). VP can also be measured in cerebrospinal fluid (CSF). Some of the stimuli which evoke VP release from the posterior pituitary may also elevate CSF VP levels. VP may play an important part in a variety of CNS functions. Substantial evidence exists implicating VP in learning and memory processes. VP may have a role in cardiovascular regulation through central pathways. Further, VP appears to act on the regulation of body temperature during fever. Other areas of central regulation where VP may be important include circadian rhythmicity, control of water intake, control of permeability of brain capillaries to water, central regulation of ACTH release and nociception. It appears that at least some of these central effects of VP involve an interaction with catecholaminergic neurotransmission.

Mammalian Bufadienolide Is Synthesized From Cholesterol in the Adrenal Cortex by a Pathway That Is Independent of Cholesterol Side-Chain Cleavage

An increasing body of evidence suggests that an endogenous mammalian bufadienolide (BD) may be involved in the regulation of Na+,K+-ATPase activity and the pathogenesis of arterial hypertension. We developed a purification scheme for marinobufagenin (MBG), an amphibian cardiotonic BD, and applied it to purify and characterize material in human plasma, culture medium conditioned by Y-1 adrenocortical cells, and rat adrenal tissue. MBG immunoreactivity purified from plasma and measured by ELISA showed important similarities (chromatography and antibody cross-reactivity) to material secreted into cell culture medium by Y-1 cells. This observation indicates that circulating mammalian BD may have an adrenocortical origin. Release of mammalian BD from adrenocortical cells grown in the absence of exogenous cholesterol was reduced by treatment of cultures with mevastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. Supplementation of the serum and cholesterol-free cell culture medium with the LDL fraction of human plasma increased the production of MBG material in the presence of mevastatin, supporting its origin from cholesterol. We used Y-1 cell lines transfected with genes shown to inhibit steroidogenesis through cholesterol side-chain cleavage (Y-1/DAX and Y-1/RIAB) to investigate the dependence of MBG biosynthesis on side-chain cleavage. Our results indicate that the mammalian BD is synthesized in the adrenal cortex from cholesterol and shares important similarities with the amphibian BD MBG, that its biosynthesis is independent of transfer of cholesterol to the side-chain cleavage enzyme complex mediated by steroidogenic acute regulatory protein, and that neither cAMP nor protein kinase A appears to be a critical component of the pathway controlling its biosynthesis.

Is ouabain an authentic endogenous mammalian substance derived from the adrenal

Ouabain has recently been reported to be an endogenous mammalian substance released by the adrenal cortex and present in normal plasma. We have attempted to confirm and extend this observation. Using a ouabain radioimmunoassay developed in this laboratory, we fractionated by high-performance liquid chromatography (HPLC) normal human plasma from healthy volunteers to determine the presence of ouabain immunoreactivity and compare this immunoreactivity with authentic ouabain. In most subjects no ouabain immunoreactivity that coeluted with authentic ouabain was observed. Some subjects had ouabain-immunoreactive material present at low levels, but it was largely attributable to cross-reactivity with diverse substances found not to be ouabain. Similar results were obtained after analysis of plasma collected from 10 patients entering a medical intensive care unit. Studies of serum-free medium conditioned by bovine adrenocortical cells showed some ouabain immunoreactivity. To determine whether this material might be a steroid product of cholesterol side-chain cleavage, we performed chemical blockade of steroidogenesis, which effectively suppressed progesterone production by these cells but had no consistent effect on ouabain immunoreactivity in this medium. Stimulation of steroidogenesis with 22-R-OH-cholesterol in bovine adrenocortical cells did not produce any increase in the ouabain immunoreactivity present in conditioned medium. Subsequent HPLC studies of ouabain immunoreactivity in bovine adrenocortical cell-conditioned medium indicated that authentic ouabain did not account for most of the ouabain immunoreactivity in serum-free medium. Studies with bovine adrenocortical cells incubated in a minimal salt and glucose medium indicated a small peak of immunoreactivity that may correspond to authentic ouabain.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of dehydration on hypothalamic control of evaporation in the cat.

1. Cats were surgically prepared with intracranial thermodes for heating of the hypothalamic thermosensitive area or with venous cannulae for measurement of blood volume and plasma osmolality. They were kept in an environmental chamber in which the ambient temperature was cycled between 25 and 38 degrees C on an 18:6 hr diurnal schedule. 2. Measurements of blood volume and plasma osmolality and of the evaporative response to hypothalamic heating were made during the 38 degrees C phase of the diurnal temperature cycle in animals when they were hydrated ad lib and in the same animals after 72‐‐96 hr of water deprivation. 3. Water deprivation produced a loss of 10% of the body weight, a significant rise in plasma osmolality and a significant fall in blood volume. 4. Hypothalamic heating in hydrated animals generated a highly significant, positive, linear relationship between hypothalamic temperature and evaporative heat loss in every case. 5. In dehydrated animals, the evaporative response to hypothalamic heating was reduced. Rates of evaporation at a given hypothalamic temperature were lower and the slopes of the lines relating evaporative heat loss to hypothalamic temperature were significantly reduced. 6. It is concluded that dehydration reduces the thermal responsiveness of central neural structures controlling evaporation in the cat.

Renal proximal tubule sodium transport and genetic mechanisms of essential hypertension

Renal sodium re-absorption is a closely regulated process serving to maintain both extracellular fluid volume and arterial blood pressure. Proteins participating in sodium re-absorption and its regulation are therefore important candidate proteins whose genes may contain sequence variation contributing to the inherited tendency for increased arterial blood pressure (essential hypertension). Important insight has come from rare forms of single–gene hypertension in human subjects and from polygenic animal models of genetic hypertension. Both indicate the primacy of altered renal function in the genesis of hypertension, and suggest that genes contributing to the disease are members of the subset of genes expressed in the kidney. This review examines evidence for abnormalities in renal sodium re-absorption in hypertension and focuses on the proximal tubule as a site of relevant dysfunction. Identification of the proteins participating in renal sodium re-absorption and its regulation, particularly those involved in the renal pressure-natriuresis mechanism, will allow gene cloning and sequencing which in turn may lead to the identification of novel gene sequence variation participating in hypertension.

REGULATION OF NA,K-ATPASE BY ENDOGENOUS OUABAIN-LIKE MATERIALS

Abstract Sodium, potassium-ATPase (NKA) is an essential energy transduction mechanism in which the free energy released by hydrolysis of ATP is transferred to an electrochemical gradient across the cell membrane. The asymmetry of sodium in this gradient is coupled to membrane transport mechanisms which affect transmembrane movement of a range of solutes and electrolytes. Recent advances in the molecular biology of NKA have revealed important new aspects of structure-function relationships as well as illuminating the basis for variations in cardiac glycoside sensitivity of the enzyme. The search for endogenous mammalian counterparts of the cardiac glycosides, which regulate the activity of the enzyme by interacting from the extracellular surface at this receptor site, has moved ahead dramatically with evidence that ouabain is an endogenous product of the mammalian adrenal cortex. These advances, and problems raised by them, are explored in this review.

Vasopressin in plasma and cerebrospinal fluid of hydrated and dehydrated steers

Levels of arginine-vasopressin (AVP), the antidiuretic hormone, have been concurrently measured by radioimmunoassay (RIA) in plasma (p) and cerebrospinal fluid (CSF) from conscious, unrestrained steers. During 4 days of dehydration, plasma osmolarity (posm) rose progressively from control hydrated levels of 301.3 +/- 0.632 mosm/1(mean +/- SE) to 338.5 +/- 3.090 mosm/1(p less than 0.001). Packed cell volume also rose from 39.9 +/- 0.64% to 44.7 +/- 1.24% (p less than 0.001). Associated with these changes was a progressive increase in pAVP from control levels of 1.3 +/- 0.19 microU/ml to 16.9 +/- 1.88 microU/ml(p less than 0.001) after 4 days without water. Log pAVP was linearly related to posm (r = 0.82, p less than 0.001). Mean level of CSF-AVP in control animals was 5.4 +/- 0.97 microU/ml. During dehydration, CSF-AVP levels also rose, becoming significantly greater than control levels (p less than 0.01) after 3 days without water and further increasing to reach 15.2 +/- 1.83 microU/ml after 4 days without water (p less than 0.001). Log CSF-AVP could also be linearly related to posm (r = 0.62, p less than 0.01). Termination of dehydration by restoration of ad libitum water supply was accompanied by return of pAVP and CSF-AVP to pre-dehydration levels. Regression analysis of concurrent levels of pAVP an CSF-AVP indicates that CSF-AVP levels are linearly correlated to pAVP levels in hydrated and dehydrated animals (r = 0.70, p less than 0.001). These experiments suggest that neurosecretory neurons secreting AVP at sites accessible to CSF respond to similar stimuli during dehydration as those neurons secreting AVP into blood.