1. It has been proposed that phenolphthalein exerts its laxative effect via an intracellular cascade that begins with the activation of nitric oxide synthase (NOS) and ends with an inhibition of NaCl and water reabsorption from the colon. Phenolphthalein also promotes the release of potassium from cells, but it is not known how this is related to its effect on sodium and water uptake. 2. An established in vitro system was used to examine the role of nitric oxide (NO) in phenolphthalein-induced release of (86)Rb(+) from COS-7 cells. 3. Sodium nitroprusside, an NOS-independent NO source, was unable to mimic the effects of phenolphthalein and N(G)-nitro-L-arginine methyl ester, an NOS inhibitor, was unable to block the effect of phenolphthalein. 4. It is concluded that NO generation is not required for phenolphthalein-stimulated potassium release. It is proposed that the effect of phenolphthalein on cellular potassium release is mechanistically distinct from the effect on NaCl and water uptake by colonocytes.

Summary1. Phenolphthalein, a well‐known laxative, stimulates the secretion of Na+ and Cl– ions and accompanying water into the intestinal tract. Measurement of 86Rb+ efflux from several, but not all, cell types indicates that phenolphthalein also results in release of cellular K+ ions.2. In the present study, the transport of 86Rb+ across human colonocyte cells (T84) cultured on trans‐well inserts was examined. The T84 cells were cultured until they developed tight junctions and a high trans‐epithelial resistance.3. Results show that phenolphthalein applied to the apical, but not the basolateral, surface of cells causes the release of 86Rb+ from the apical surface. Basolateral treatment of cells with phenolphthalein had no effect on the release of 86Rb+.4. Simultaneously with the increased 86Rb+ efflux, indirect evidence of enhanced Na+/K+‐ATPase activity was also observed.5. Although ouabain inhibited the increased Na+ pump activity, it did not affect apical 86Rb+ release.6. As evidenced by near steady state 86Rb+ uptake data, the increased Na+/K+‐ATPase activity was insufficient to restore intracellular concentrations of K+ in the presence of phenolphthalein.7. 4,4(9‐Fluorenylidene)diphenol, a homologue of phenolphthalein, had a similar effect on 86Rb+ transport by T84 cells.8. These results indicate a primary stimulation of 86Rb+ efflux from the apical surface of polarized T84 cells by apically applied bis‐phenolic compounds.9. A secondary stimulation of the basolateral Na+/K+‐ATPase is thought to result from intracellular Na+ increase, as documented in several other cell types exposed to bis‐phenolic compounds, although not directly measured in these experiments.10. The results also indicate that bis‐phenolic compounds interact specifically with some apical but not basolateral membrane structures in regulating 86Rb+ efflux from polarized T84 cells.

Used worldwide since 1980 for the prevention of breast engorgement in the puerperium, in 1994 bromocriptine mesylate was withdrawn from the American market as an agent suitable for ablactation. The relevant recommendation of the Food and Drug Administration rested on case reports that described severe vasospastic reactions among users of the drug. Some patients so affected suffered stroke, intracranial bleeding, cerebral edema, convulsions, myocardial infarction, and puerperal psychosis. More recently, it has been suggested that the side effects of the drug may also include circulatory collapse secondary to cardiac dysrhythmia. This report describes two additional cases in this category. The antepartum clinical evaluation of these women suggested that they were predisposed to arrhythmias.

The laxative action of phenolphthalein (5) is believed to result from induction of potassium and water efflux from the colon epithelium. In cultured cells, K+ efflux is promoted by 5 and by a contaminant (1) present in commercial phenol red. Six compounds with chemical structures related to those of 5 and 1 were tested for ability to induce the release of 86Rb from COS-7 cells preloaded with this isotope: 4,4'-(9-fluorenylidene)diphenol (2), 4, 4'-(9-fluorenylidene)dianiline, 4, 4'-(9-fluorenylidene)bisphenoxyethanol, 1,1'-bi-2-naphthol, 4, 4'-biphenol, and bis(4-hydroxyphenyl)methane. With one exception these compounds were all inactive at a concentration of 10 microM. However, 2 caused profound 86Rb efflux at concentrations as low as 100 nM. Concentrations of 5 1-2 orders of magnitude higher were needed to achieve similar levels of activity. The three compounds known to be active in this experimental system share a common feature that is absent in all the inactive compounds: a five-membered ring structure, one of whose carbon atoms is disubstituted with p-hydroxyphenyl residues. Because 2 and 5 are readily available, comparative studies on the mechanism of action of these biphenols at the cellular level can now be undertaken.

Epstein-Barr virus-transformed lymphoblasts from patients with essential hypertension demonstrate enhanced G protein-mediated cytosolic free calcium ([Ca2+]i) response to platelet-activating factor (PAF). To map genes responsible for variation in G protein-coupled signaling, we used this cellular phenotype for a linkage study of transformed cell lines from the Centre d'Etude du Polymorphisme Humain (CEPH) reference pedigrees. The PAF-evoked change in [Ca2+]i ranged from 20 to 392 mmol/L and was highly reproducible within each cell line. PAF-elicited [Ca2+]i responses were obtained in lymphoblastic cell lines from five densely mapped pedigrees of the CEPH collection. Using PAF-evoked [Ca2+]i responses as a quantitative trait, two-point sibpair linkage analyses were conducted using 5150 markers from the Collaborative Human Linkage Center (CHLC) database. Nine loci, located on chromosomes 1, 4, 10, 11, 13, 16, and 17, were suggestive of linkage, with values of P < 7.4 x 10(-4). Multipoint linkage analysis produced a significant linkage finding (P = 2.1 x 10(-5) in one family at D16S151, with suggestive linkage results for seven additional markers spanning a 40-cM interval of chromosome 16. Multipoint analysis produced suggestive findings of linkage to eight loci from two distinct regions of chromosome 11 in another family. These results indicate that loci involved in the control of G protein-mediated mechanisms, suggested to be involved in the pathophysiology of essential hypertension, can be identified using cell lines from general pedigrees selected without any knowledge of the blood pressure status of the donors. This strategy represents an approach to rapidly and inexpensively mapping loci related to common, complex disorders, using phenotypes that are stable in immortalized lymphoblasts together with existing reference pedigree cell lines and genotype databases.

In order to further characterize previously described racial differences in intracellular Ca2+ homeostasis, the activity of sarcoplasmic-endoplasmic reticulum Ca2+-ATPase was measured in cultured skin fibroblasts from normotensive African-Americans and whites. Cells originated from apparently healthy, normotensive African-American (n = 15) and white (n = 15) subjects. The activity of the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase was measured by: 1) oxalate-dependent only, and 2) oxalate-dependent, thapsigargin-sensitive 45Ca2+ uptake of membrane homogenates. Over 90% of the oxalate-dependent 45Ca2+ uptake was thapsigargin-sensitive. There was no detectable racial difference in the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase activity with either technique. It is concluded that the previously described higher cellular Ca2+ turnover and larger cellular stores of exchangeable Ca2+ in cells from African-Americans versus whites are not due to changes in the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase itself. The findings do not exclude possible increased activity of this enzyme in intact cells of African-Americans where regulatory mechanisms not present in the membrane preparation may operate.

Evidence for the involvement of endothelial cells in the pathogenesis or erythropoietin-induced hypertension, and for endothelial cell damage in patients with chronic renal failure, has emerged and appears to be of major concern. We, therefore, investigated the effect of recombinant human erythropoietin (rHuEPO) therapy on endothelium-derived hormones in predialysis patients with progressive renal anemia. At the entry to the trial, the serum thrombomodulin concentration (Tm) and plasma endothelin-1 concentration (ET-1) in the predialysis patients were significantly higher than those in age- and sex-matched normal subjects. Following a 16 week period of treatment with 6000IU rHuEPO given intravenously once a week, patients' hematocrit increased from 27.1 +/- 2.6% to 34.6 +/- 3.2% (n = 16, P < .001). A positive correlation was found between Tm and serum creatinine concentration (Cr) (r = 0.61, P < .05 (n = 16), but no correlation was found between ET-1 and Cr. Tm and Tm/Cr significantly decreased from 7.9 +/- 2.8 ng/mL to 6.6 +/- 2.4 ng/mL (P < .01, n = 16), and from 2.1 +/- 0.7 (x10(-10) to 1.6 +/- 0.7 (x10(-10), P < .01, n = 16), respectively. However, there was no change in ET-1 as a result of the rHuEPO therapy. Creatinine clearance (Ccr), Cr, total amount of daily Tm excretion, Tm clearance/Ccr, daily urinary protein and albumin excretion, and blood pressure also remained unchanged throughout the trail. The present study indicates that correcting anemia by rHuEPO therapy reduces an abnormally elevated Tm in predialysis patients while blood pressure and renal function remain unchanged, suggesting that rHuEPO has a beneficial effect on endothelial cell dysfunction in chronic renal failure patients. This effect may be mediated via an improved oxygen supply to the endothelial cells due to the amelioration of anemia by rHuEPO.

Three cases of myocardial infarction (MI) in women taking bromocriptine for milk suppression are presented. The incidents occurred in 1993 and 1994, the last two only a few weeks before the withdrawal of the drug from the American market as a drug suitable for ablactation. In one patient, the MI presented on the 12th day postpartum and was accompanied by signs and symptoms reminiscent to ergotism. Another mother suffered MI, accompanied by hypertension, six days after a vaginal delivery complicated by postpartum haemorrhage. The third patient began to take bromocriptine more than 2 weeks postpartum and died suddenly 24 days after her childbirth. To the knowledge of the authors, these are the 12th to 14th Literary reports describing an apparent association between the use of bromocriptine for ablactation and the occurrence of MI in the puerperium.

A 34-year-old mother developed acute myocardial infarction (MI) 10 days postpartum while taking bromocriptine for ablactation. Her only known risk factor was moderate hypercholesterolemia. The initial coronary arteriogram revealed minimal irregularities of the mid- and distal left anterior descending (LAD) artery, but severe narrowing of a large first diagonal branch of the LAD. Repeat coronary arteriography, performed two months later for recurrent chest pain, showed no significant change in the LAD. There was, however, a marked improvement in the first diagonal branch. To the authors' knowledge this is the 10th report of MI in a patient using bromocriptine postpartum for milk suppression and only the second one to implicate by its investigations the role of vasospasm in the coronary vessels at the cause of the infarction.

Recent literary case reports indicate that bromocriptine mesylate, when used for the suppression of lactation in the puerperium, can cause serious and even lethal side effects. The untoward sequelae are attributed to generalized or focal vasospasm affecting the cardiac and/or cerebral bold vessels. Apart from pre-existing hypertension and use in association with other ergot derivatives, the factors predisposing to such complications have not been elucidated, The authors present three atypical bromocriptine related postpartum accidents which may expand the understanding both of the predisposing factors and the potential consequences of bromocriptine related severe side effects. One of the cases raises the suspicion that the manifestations of hyperthyroidism may be aggravated by this method of pharmacologic ablactation. Another observation appears to imply that the drug may trigger the onset of chronic hypertension in women so predisposed. The development of cerebral infarcts, identified by MRI, in a clinically asymptomatic woman, exemplifies the threat of recurrent seizure activity in cases of bromocriptine related stroke.

An impurity of phenol red (PRI) has been shown to markedly alter the intracellular Na+ and K+ homeostasis of several cell types. The effect of PRI seems to involve intracellular Ca(++)-dependent mechanisms. Using COS-7 cells as a model, we further characterized the mechanism of action of PRI by measuring cellular Na+/K+ contents and 86Rb+ efflux. Similar to human skin fibroblasts, in COS-7 cells calmodulin inhibition moderated the cationic transport effects of PRI. A TMB-8 dependent intracellular Ca++ pool does not seem to be involved in these transport events. We found no evidence for participation of the transcriptional-translational machinery in the effect of PRI. Both quinine and quinidine are able to prevent nearly all changes caused by PRI in the cellular Na+/K+ contents and 86Rb+ efflux. Although phenol red contained multiple impurities by high performance liquid chromatography (HPLC), phenolphthalein, a structurally close relative of phenol red, was free of any detectable contamination. Phenolphthalein elicited qualitatively similar transport changes to those observed during exposure to PRI. Regardless of the exact mechanism of action, we propose that the as yet unidentified substance is not a cellular toxin, rather it is a cationic transport modulator. Directly or indirectly, it may interact with the cellular Ca++/calmodulin system and activate some quinine/quinidine sensitive transport processes. This transport process is likely to be a Ca(++)-sensitive K+ channel but, due to the lack of specificity of quinine and quinidine, other transport mechanisms must be also considered. The chemical nature of PRI may be similar to phenolphthalein.

Previously, we described substantial alterations in the Na+ and K+ homeostasis of human skin fibroblasts following removal of fetal bovine serum (FBS). Herein, we report that FBS removal per se does not cause any cellular ionic changes unless a lipophilic impurity of commercial phenol red preparations is present. This substance accelerates Rb-86+ efflux four to seven times, causes a four to eight time increase in cellular Na+, and a 40-70% reduction in cellular K+ contents. FBS (10%) or albumin (0.8%) appears to bind the impurity thus inhibiting its action. The increased cellular Na+ and decreased K+ contents do not return to baseline within 4 hours following the removal of the phenol red extract. However, albumin completely reverses the cellular cationic changes that develop during a 2 hour exposure of the cells to the free substance. The reversibility of its action by albumin suggests that the substance exerts its effect on or within the cell membrane and not intracellularly. Among seven different cell lines tested the Rb-86+ eff lux from, and the Na+-K+ contents of, COS-7 and Hs68 cells also responded to unpurified phenol red in a way similar to human fibroblasts. The amount of the phenol red contaminant is manufacturer dependent. As little as 0.5 muM phenol red, from one vendor, was sufficient to elicit response in the Rb-86+ efflux. Given that the impurity is unlikely to be more than a small fraction of phenol red, it seems to be a potent ionic transport modulator. Based on these results, the presence of commercial phenol red in serum-free growth or test media, including the increasing variety of chemically defined culture media, should be considered as a potential confounding factor in measurements that depend on intracellular Na+-K+ homeostasis. The findings of such earlier studies may need to be reconsidered if the cells were exposed to unbound phenol red. We recommend that, until the manufacturers further refine their product, phenol red be purified by ether extraction before its use. The evaluation of the potential physiologic or pharmacologic relevance of this potent cation transport modulator awaits its isolation. (C) 1993 Wiley-Liss, Inc.

The relationships of sitting heights and body weights of fetal twin pairs were analyzed in comparison with established growth rate standards of singleton fetuses. The apparent rate of growth of individual twins scattered around the average growth curve in the same manner as singletons. In contrast, members of 12 twin pairs with <125 mm sitting heights, were closely similar in terms of bodily dimensions. Among the 7 pairs with crown-rump lengths of >125 mm, 4 were discordant, suggesting a deceleration of growth in one of the twin fetuses, starting after the 18th week of gestation.

Previously, we demonstrated that removal of fetal bovine serum (FBS) from the medium of human skin fibroblasts resulted in an accelerated 86Rb+ washout, decreased cellular K+, and increased Na+ contents. In the present study we examined the mechanism underlying these changes. The efflux rate constant for 86Rb+, and the cellular contents of Na+ and K+ were measured. Verapamil (K1/2 = 15 microM) and chlorpromazine (K1/2 = 1 microM) reduced by approximately 70% the increased 86Rb+ washout evoked by FBS removal. The effect of the two drugs was additive at low, but not high, concentrations. Verapamil and chlorpromazine also attenuated the decrease in cellular K+ content and prevented the increase in cellular Na+ content associated with FBS depletion. Bumetanide (50 microM) was only partially effective in offsetting the enhanced 86Rb+ efflux and was completely without any effect on the cellular Na+ and K+ changes induced by FBS removal. In the presence of FBS, A-23187 produced a slight and transient increase of the 86Rb+ washout. The protein kinase C activator phorbol 12-myristate 13-acetate enhanced the 86Rb+ efflux in FBS-containing medium for a prolonged period but this increase was only a fraction of that caused by serum removal. Cellular Na+ and K+ contents were not changed by the phorbol ester. We conclude that FBS removal raises the cellular Na+ content, and enhances 86Rb+ efflux, through a calmodulin-dependent pathway activated by calcium influx.

The study presents an overview of the changes in perinatal mortality rates at the Statewide Perinatal Center of New Jersey during the past decades. According to the data, the increase in the rate of cesarean sections from 4.5 percent to 17 percent, and the comparable reduction of the rates of manipulative intrapartum and extraction procedures, contributed significantly to the decrease of the perinatal mortality rates from 51/1000 to 17/1000 between 1971 and 1983. Of the new technical tools, those utilized for the evaluation of fetal well-being antepartum appeared to be more useful then those used intrapartum. On account of the high prevalence of genital infections in the population, the recent acceptance in the service of the use of invasive intrapartum technology, appears to have impacted unfavorably upon the perinatal mortality trends. The increased rate of births of premature babies, the widespread abuse of habit forming drugs in the community, and the routine use of procedures requiring artificial rupture of the membranes, probably all contributed to the rapid increase of the perinatal mortality rate in the Center from 15/1000 in 1986 to 28/1000 in 1988. It is concluded that perinatal care is a complex medical and social task. The overall result of the relevant efforts depends to a great extent upon the social environment, and the moral standing, educational level and motivation of the recipients.