Evaluation of the tilt test in an adult emergency medicine population.

STUDY OBJECTIVE: To evaluate the usefulness of the tilt test in identifying significantly dehydrated or occultly bleeding adult patients in an emergency department. DESIGN: Prospective. SETTING: Urban ED. TYPES OF PARTICIPANTS: Two hundred two ill adult patients with complaints suggestive of dehydration and/or occult blood loss and a control sample of 21 healthy individuals. INTERVENTION: Orthostatic measurements were taken from blood samples from the ill and healthy patients. The estimates of the degree of dehydration measured or calculated were total body water deficit percentage (based on measured serum osmolality), ratio of blood urea nitrogen to creatinine, creatinine, and hematocrit. Multiple historical and physical examination variables related to dehydration or occult bleeding were recorded. MEASUREMENTS AND MAIN RESULTS: Multiple analysis of variance testing of the historical and examination variables in the 166 dehydrated patients revealed the absence of syncope (P = .037) and the lack of axillary sweat (P = .026) to be significantly associated with an increased level of dehydration. Regression analysis of the orthostatic measurements and age with the estimates of dehydration found that orthostatic change in heart rate (P = .0165) and age (P = .0047) demonstrated a very small (r2 = .098) association with the level of dehydration. Orthostatic changes in systolic blood pressure and diastolic blood pressure demonstrated no statistically significant association with level of dehydration. There was wide variation in the tilt test measurements in samples from both the healthy and the ill patients: heart rate, 6.8 +/- 7.8 versus 13.2 +/- 10.5; systolic blood pressure, -2.5 +/- 8.0 versus -8.3 +/- 12.8 mm Hg; and diastolic blood pressure, 5.3 +/- 9.9 versus 0.6 +/- 8.6 mm Hg, respectively. The only parameter that attained statistical significance between the 36 blood-loss patients and the healthy patients was change in systolic blood pressure (P = .001); however, this was not clinically significant (-10.7 +/- 13.7 versus -2.5 +/- 8.0 mm Hg, respectively). CONCLUSION: It appears impossible to define a "positive" tilt test that would adequately identify patients with clinically significant dehydration or blood loss; this is due to the large variance in patients' orthostatic measurements both in a healthy and in an ill state and the lack of a significant correlation of orthostatic measurements to a level of dehydration. Lack of axillary sweat, no complaint of syncope, and younger age all indicate greater degrees of dehydration.

Unusual subclavian artery lacerations resulting from lap-shoulder seatbelt trauma: case reports.

While the impact of seatbelts on injury is favorable, their use is also associated with unique and previously unrecognized patterns of injury. Two cases of combined subclavian and vertebral artery injury, when lap shoulder harness restraints were used, are presented.

Spontaneous rupture of the spleen: a case report and literature review.

Traumatic rupture of the spleen has been well described in the medical literature and is found in approximately 30% of patients undergoing surgery for blunt abdominal injury. Atraumatic splenic rupture is described much less often. A case of atraumatic rupture of an enlarged spleen is described. The etiology of spontaneous rupture of the spleen in this case could not be determined. The patient was seen and discharged twice from the emergency department; the diagnosis was made at emergency laparotomy three days after subsequent admission. This case should remind the emergency physician that nontraumatic splenic rupture should be considered in the differential diagnosis of unexplained acute left upper quadrant abdominal pain.

The effect of tracheostomy on posterior cricoarytenoid muscle.

It has been reported previously that the amount of electromyographic (EMG) potential of the posterior cricoarytenoid (PCA) decreases after prolonged tracheostomy. It is, therefore, reasonable to assume that a significant alteration of the biochemical characteristics of this muscle would also occur. In addition to histochemical analysis, endoscopic and EMG data were recorded to give a direct comparison in each subject. Seven male beagles were used for this study. Four were tracheostomized and three served as controls. They were examined immediately before and after surgery and again after 4 weeks by EMG and endoscopic techniques. Histochemical staining was performed on each subject. All three modalities failed to demonstrate a substantial difference between the controls and the experimental dogs.

Structure-activity relationships of estrogens.

The last 50 years has seen an exponential rise in the published reports about estrogen action. The model to describe the early events in the mechanism of action of estrogens via the estrogen receptor is updated in this paper to incorporate some of the recent data on the subcellular localization of the receptor. New evidence suggests that the receptor is a nuclear protein, so it appears that estrogens must first diffuse into the nuclear compartment to initiate estrogen action via the receptor complex. This review traces the development of potent estrogenic compounds by the study of their structure-activity relationships. Studies of structure-activity relationships in vivo using Allen Doisy or 3-day uterine weight tests can provide much valuable information, but the assays suffer from the complex problems of pharmacokinetics and metabolic transformation. Studies in vitro using primary cultures of rat pituitary or uterine cells to assay the ability of a compound to induce prolactin synthesis or progesterone receptor synthesis, respectively, can provide essential information about the structural requirements for a compound to produce estrogenic effects. Nevertheless, it should be pointed out that studies in vivo are required to determine whether a compound is metabolically activated to an estrogen. Estrogen receptor binding models are presented to describe the changes in a molecule that will predict high affinity for the ligand and agonist, partial agonist and antagonist properties of the ligand-receptor complex. Most estrogenic pesticides and phytoestrogens comform to the predictions of the estrogen receptor binding model.

Estrogen-stimulated prolactin synthesis in vitro. Classification of agonist, partial agonist, and antagonist actions based on structure.

An in vitro assay that depends upon the synthesis of prolactin by primary cultures of dispersed cells from immature rat pituitary cells was used to study the structural requirement for estrogen action. Two categories of estrogens were identified: full estrogens (agonists) and partial estrogens (partial agonists) with antiestrogenic actions against the effects of 0.1 nM estradiol (E2). All of the agonists [diethylstilbestrol (DES), dimethylstilbestrol (DMS), R2858, and RU16117] produced a dose-related increase in prolactin synthesis equivalent to E2, although potencies were different: E2 = DES = R2858 greater than RU16117 greater than DMS. Partial agonists [ICI 3188, tri(4-hydroxyphenyl)chloroethylene, and bisphenol] each had bis(4-hydroxyphenol) substitutions at the ethylene double bond and stimulated prolactin synthesis only to about 50% of the maximal response observed with E2. Trianisylchloroethylene was weakly active as a partial agonist, but at high concentration (10 microM) was able to decrease prolactin synthesis produced by 0.1 nM E2. Previous studies from these laboratories showed that triphenylethylene derivatives with a strategically located alkyl aminoethoxyside chain are complete E2 antagonists with no agonist activity. Two series of novel compounds were assayed to determine whether their structures would predict biological activity. LN2299, the cis geometric isomer of a triphenylbromethylene, was a full agonist with activity equivalent to zuclomiphene, the cis geometric isomer of clomiphene. Cyclofenyl was a partial agonist, but deacetylation to the diphenol increased partial agonist activity and potency. However, introduction of a single pyrrolidinoethylside chain into the deacetylated cyclofenyl increased antiestrogenic potency and completely suppressed the expression of agonist activity. Finally, LN2833, with a novel C(OH)CH3 side chain in the position normally occupied by the alkylaminoethoxyside chain of most antiestrogens, produced antiestrogen activity with no estrogen properties. Antiestrogenic potency was increased in LN2839 by a phenol in the triphenylethylene in a position equivalent to the 3-phenolic hydroxyl of E2. In general, the potency and biological properties could be predicted by reference to the structure of the molecule. Potent estrogens or antiestrogens have a phenolic hydroxyl in a position that would be equivalent to the 3-phenolic hydroxyl of E2. Partial agonist action is predicted by a 4-hydroxyphenol attached to the same carbon as the phenyl ring equivalent to the A-ring of E2.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural requirements for the pharmacological activity of nonsteroidal antiestrogens in vitro.

The structure-activity relationships of a tamoxifen (TAM) (Z-1-(4- beta-dimethylaminoethoxyphenyl)1,2-diphenylbut-1-ene) series have been investigated. The tamoxifen derivatives were assayed in vitro by their modulation of estradiol (E2)-stimulated prolactin synthesis in primary cultures of dispersed rat pituitary gland cells. Monohydroxylation of TAM in position 4 of the stilbene ring system was found to be the optimal substitution for binding to the estrogen receptor [relative binding affinity (RBA) = 234] and to inhibit E2 (1 nM)-stimulated prolactin synthesis (IC50 7 nM) by pituitary cells in primary culture. Substitution in positions 3 and 4 to form a catechol did not decrease affinity for the estrogen receptor (RBA = 252), and potency as an antiestrogen was maintained in the prolactin assay (IC50 20 nM) as long as oxidation of the catechol was prevented. All of the hydroxylated derivatives of tamoxifen tested were estrogen antagonists; however, removal of the alkylaminoethoxy side chain from TAM produced a full estrogen agonist with low potency (20 nM). In contrast, removal of the side chain from 4-hydroxytamoxifen (4-OH TAM) produced a partial agonist. A structural analogue of 4-OH TAM, 3-[beta-dimethylaminoethoxy]-11-ethyl-12-(4-hydroxyphenyl)5,6- dihydrodibenzo[a,e]-cyclooctene (7c) had a decreased potency (IC50 16 nM) compared with 4-OH TAM (IC50 4 nM in the same experiment) as an estrogen antagonist. If the side chain was changed from a dimethylaminoethoxy to glyceryl, antagonist activity was reduced (IC50 0.8 microM). An allyl side chain produced a compound with no antiestrogenic activity at concentrations up to 1 microM. An adaptation of Belleau's macromolecular perturbation theory is suggested to explain the interaction of agonists, antagonists, and partial agonists at the ligand binding site of the estrogen receptor.

Nuclear localization of unoccupied oestrogen receptors.

According to the current model of steroid hormone action oestrogen is thought to bind to its receptor in the cytoplasm of target cells and the oestrogen-receptor complex is then translocated into the nucleus. This model is based on evidence obtained in homogenized cell preparations in which free receptor is associated with the cytosol, whereas steroid-bound receptor is associated with the nuclear fraction. Some data suggest, however, that the unfilled receptor may reside in the nucleus, and that cytosolic localization represents an extraction artefact. We have now reinvestigated the subcellular distribution of unfilled oestrogen receptor using cytochalasin B-induced enucleation to obtain cytoplast and nucleoplast fractions from receptor-containing GH3 cells derived from rat pituitary tumours. We found that cytoplasts prepared from GH3 cells contain little oestrogen-binding activity and that most of the unfilled oestrogen receptors are associated with the nuclear fraction. We therefore suggest that the standard model is in error and that the unoccupied receptor is nuclear in the intact cell.

The inhibition of prolactin synthesis in GH3 rat pituitary tumor cells by monohydroxytamoxifen is associated with changes in the properties of the estrogen receptor.

E2 (1 nM) stimulated the synthesis of PRL in GH3 cells. OH TAM (100 nM) did not affect basal PRL synthesis, but completely inhibited the increase produced by 1 nM E2. [3H]E2 and [3H]OH TAM both bound to the cytosolic 8S ER and these were split into 4S subunits on sucrose gradients containing 0.4 M KCl. By comparison, ER complexes extracted from nuclei of GH3 cells cultured in media containing [3H]E2 or [3H]OH TAM both sedimented at 5S on sucrose gradients containing 0.4 M KCl. Both 4S and 5S ER complexes were recognized by the monoclonal antibody D547 which increased their sedimentation coefficients to 8-9S. In contrast, a polyclonal antibody raised to calf uterine ER in the goat, interacted with the cytosolic ER so that the binding of [3H]E2 was inhibited but the binding of [3]OH TAM was only slightly reduced. A molecular model is proposed to describe the binding of E2 and OH TAM to the ER that might contribute to an understanding of estrogen and antiestrogen action.

8. Steroids and differentiation. The role of estrogen in the differentiation of prolactin producing cells.

The capacity to synthesize PRL and GH was studied in normal mice and in Snell and Ames dwarfs. In normal mice GH synthesis turned on dramatically between day 16-17 of gestation whereas PRL was not detectable throughout gestation or the first week of life, was barely detectable in 8-day-old mice and was clearly demonstrable by 12 days of age. However, transplantation of pituitaries from newborn mice into adult female hosts resulted in substantial PRL synthesis. Treatment of mice with DES at various ages showed that neonates failed to respond to the hormone; in older pups, an age-dependent increase in the magnitude of PRL synthesis was observed. There was a direct correlation between nuclear estrogen receptor levels and PRL cell function. Snell and Ames dwarf mice failed to synthesize PRL or GH at any stage of development, nor could we detect immunoreactive peptides which might represent mutant forms of the hormones. Our findings suggest that: (1) GH gene expression precedes that of PRL by about 2 weeks; (2) the development of PRL cell function is dependent on estrogen; (3) the capacity to respond to estrogen is present before the endogenous initiation of PRL synthesis and is limited by the availability of estrogen receptor; and (4) in Snell and Ames mutants, both types of alleles result in failure of the pituitary to initiate PRL and GH synthesis.

Direct and reversible inhibition of estradiol-stimulated prolactin synthesis by antiestrogens in vitro.

The antiestrogens tamoxifen and monohydroxytamoxifen inhibited the estradiol-stimulated increase in prolactin synthesis by dispersed cells in culture derived from immature rat pituitary glands. Monohydroxytamoxifen had a relative binding affinity for the estrogen receptor similar to that of estradiol, whereas tamoxifen's relative binding affinity was approximately 3%. This was consistent with the observation that monohydroxytamoxifen was 30 times more potent than tamoxifen as an antiestrogen in vitro. To avoid the possibility that tamoxifen was fractionally metabolized to monohydroxytamoxifen by the pituitary cells, the p-methyl, p-chloro, and p-fluoro derivatives of tamoxifen that are unlikely to be converted to monohydroxytamoxifen were tested for activity. The substitution did not have a detrimental effect on their ability to inhibit the binding of [3H]estradiol to either rat uterine or pituitary gland estrogen receptors. Similarly, the derivatives of tamoxifen inhibited estradiol-stimulated prolactin synthesis at concentrations that were consistent with their relative binding affinities. Although it is clearly an advantage for tamoxifen to be metabolized to the more potent antiestrogen monohydroxytamoxifen, we have shown that this is not a prerequisite for the antiestrogenic actions of tamoxifen. With the direct actions of antiestrogens established, the pituitary cell system was validated for further structure-activity relationship studies. Overall, the inhibition of estradiol-stimulated prolactin synthesis by antiestrogens is competitive and reversible with estradiol, an effect that can be explained by interactions with the estrogen receptor system.

An estrogen receptor model to describe the regulation of prolactin synthesis by antiestrogens in vitro.

A hypothetical model of the ligand interaction with the estrogen receptor binding site has been developed to describe the structural features necessary to initiate or to inhibit prolactin synthesis in vitro. The biological potency of the binding ligands is directly related to their relative binding affinity (RBA) for the estrogen receptor. The relative potencies of antiestrogens to inhibit estradiol-stimulated prolactin synthesis was trans-monohydroxytamoxifen identical to cis-monohydroxytamoxifen identical to tamoxifen, consistent with their RBAs for uterine estrogen receptor. Similarly the relative potency of estrogens to stimulate prolactin synthesis was diethylstilbestrol identical to estradiol greater than ICI 77,949 greater than ICI 47,699 identical to zuclomiphene, consistent with their RBAs. The compound LY126412 (trioxifene without the aminoethoxy side chain) did not interact with the estrogen receptor at the concentrations tested (10(-8)--10(-6) M) or exhibit estrogenic or antiestrogenic properties using the prolactin synthesis assay. Overall, the ligand-receptor model stresses the structural requirement for high affinity binding and the critical positioning of the alkylamino-ethoxy side chain in space (in relation to the ligand-binding site on the estrogen receptor) to prevent prolactin synthesis.

Genomic organization of prolactin and growth hormone coding sequences in dwarf and normal mice.

The genomic organization of DNA encoding growth hormone (GH) and prolactin (PRL) genes has been investigated in two types of homozygous dwarf mice and their normal counterparts. We have previously shown that dwarf mice of either strain fail to initiate pituitary synthesis of GH or PRL during perinatal development. Analysis by Southern transfer of restriction enzyme-digested DNA and hybridization to 32P-labeled cloned probes derived from bovine GH and rat PRL mRNAs revealed no evidence for deletions or rearrangements in or around the structural genes for GH or PRL in either dwarf genotype. In situ hybridization of the probes to pituitary and liver tissue slices failed to detect specifically hybridizing RNA species in dwarf pituitaries. These findings suggest that the pleiotropic effects of the dwarf mutations, which lead to abnormalities in transcription of the genes or in the processing of transcripts, may be due to blocks in the development of functional somatotrophs and mammotrophs.

Evidence that autoregulation of prolactin production does not occur at the pituitary level.

PRL production in primary cultures of rat pituitary cells was examined for evidence of autoregulation. The kinetics of PRL accumulation in the culture medium were linear (r = 0.993) over a 20-fold range of PRL concentrations (85-1800 ng/ml). The addition of 0.01-1000 ng/ml biologically active ovine PRL for 2 h had no effect on the rate of secretion or the intracellular levels of PRL. The functional integrity of the cells was verified by the demonstration that estradiol (10(-8) M) caused a 3-fold increase in both PRL synthesis and secretion. Ergocryptine (10(-8) M) inhibited PRL secretion by 57% (P less than 0.001), and this response was modulated by pretreatment with estradiol. Increasing concentrations of endogenously secreted PRL did not detectably alter the rate of incorporation of [35S]methionine into cellular PRL in control or ergocryptine-treated cells for up to 8 h. These results indicate that autoregulation of PRL does not operate directly at the level of the pituitary.

Prolactin synthesis in primary cultures of pituitary cells: regulation by estradiol.

Pituitary cells cultured with estradiol respond by a specific increase in prolactin synthesis. Extensive inhibition of DNA synthesis (61-78%) with hydroxyurea or cytosine arabinoside resulted in only 28-33% decrease in estrogen-induced prolactin synthesis. To assess the role of prolactin cell proliferation in the estrogen-induced response, mammotrophs were identified by immunocytochemistry. Cultures treated with estradiol for 1, 2 or 5 days contained 101 +/- 1, 113 +/- 2 and 132 +/- 1% of the number of mammotrophs in controls. Estradiol treatment for corresponding periods resulted in prolactin synthesis representing 94 +/- 5, 144 +/- 11 and 270 +/- 22% of controls and prolactin mRNA levels representing 115 +/- 7, 160 +/- 7 and 322 +/- 22% of controls. Thus estrogen caused a considerable increase in prolactin synthesis which paralleled the increase in prolactin mRNA levels and a much smaller relative increase in the number of mammotrophs. We conclude that regulation of prolactin synthesis by estrogen is mediated predominantly but not exclusively through stimulation of gene expression in existing pituitary cells.

Growth hormone and prolactin synthesis in normal and homozygous Snell and Ames dwarf mice.

The severe growth retardation and sterility characteristic of Snell and Ames dwarfs, two nonallelic, recessive mouse mutants, have been attributed to a deficiency in pituitary production of GH and PRL. We have investigated the synthesis of these hormones in normal and homozygous dwarf mice of both strains at various stage of development to determine whether the mutations prevent initial development of the pituitary capacity to produce these hormones. Synthesis of radiolabeled GH and PRL was assayed by two-dimensional electrophoresis and immunoprecipitation using goat antisera to mouse GH and PRL. Litters in which all pups were mutant were produced by mating adult dwarfs made fertile by implanting a normal pituitary under the kidney capsule. We found that GH and PRL synthesis was undetectable in Snell or Ames dwarfs at all stages of development examined, including the neonatal period when dwarf pups are indistinguishable from normal littermates. Furthermore, we detected no immunoprecipitable peptides which might represent mutant hormones in these animals. Assays of GH synthetic rates in heterozygous animals indicated that there may be a slight negative effect of the mutant gene on GH synthesis in Snell animals. It was concluded that in the homozygous condition, both types of dwarf alleles result in failure of the pituitary to initiate GH and PRL synthesis.

Regulation of pituitary growth and prolactin gene expression by estrogen.

We presented evidence that primary cultures of rat pituitary cells respond to estradiol by increased incorporation of precursors into prolactin. The response is specific for estrogenic hormones and is maximal at physiological concentrations of estradiol. The time course and magnitude of the response in cultured cells is in agreement with that observed under in vivo conditions, suggesting that estrogen exerts its effect mainly through a direct action on the pituitary gland. The data presented indicate that estrogen stimulates prolactin synthesis predominantly through increased prolactin mRNA accumulation, and to a lesser extent, through mammotroph cell proliferation. Chronic treatment with DES caused sustained proliferation of pituitary cells leading to prolactin producing pituitary tumors in the Fischer 344 rat, but not in the Holtzman rat. The genetic basis for these differences are currently under investigation.