Experimental Biology and Medicine

I. Introduction

In the autumn of 1979, three independent laboratories (1–3) described the chemical structure of a new subclass of phospholipids that possessed biologic properties identical to platelet-activating factor (PAF) and an antihypertensive polar renal lipid (APRL). The chemical features of this unique bioactive phospholipid are an O-alkyl ether group at the sn-1 position, an acetate at the sn-2 position, and a phosphocholine moiety at the sn-3 position of the sn-glycerol moiety (Fig. 1). A number of reviews have provided a detailed account of earlier developments in the PAF (4–11) and APRL (10, 12) fields and, therefore, only significant historical events are highlighted in this section.

The antihypertensive activity of an extract from the renal medulla was originally observed in dogs with renoprival hypertension (13). This antihypertensive principle could also be extracted from renomedullary interstitial cells of rats and was initially referred to as ARH, an antihypertensive renomedullary hormone (14). However, further characterization of the polar nature of this lipid led to changing the name of ARH to APRL, the abbreviation for antihypertensive polar renomedullary lipid (15).

Within the same time frame of the APRL investigations, several articles (16–18) appeared which described the release of vasoactive amines from rabbit platelets in a reaction that required leukocytes from an immunized rabbit, an antigen, and platelets; the reaction, referred to as leukocyte-dependent release of histamine (LDHR), was considered as an immediate hypersensitivity-type reaction linked to allergic responses. Benveniste et al. (19) further characterized this soluble factor and identified IgE-stimulated basophils as the source of the substance responsible for LDHR and the aggregation of platelets; these workers called the bioactive substance PAF. The lipid nature and polar properties of PAF finally became apparent from chemical studies of its solubility characteristics, chromatographic behavior on silicic acid, and reactivity with lipases (20, 21).

Primary cultures of renal cortical cells prepared by selective sieves have been found to display some characteristics of renal proximal tubular epithelium but their site of origin has not been confirmed by electrophysiologic studies. Cells were cultured in a defined medium on collagen gels. Confluency was approached after 7–10 days but gels were found to have zero transepithelial resistance unless they were allowed to contract spontaneously. With the appearance of a nonzero resistance, there was a change in morphology to a more columnar cell with better developed microvilli. These structural features were particularly prominent in clusters of proliferating cells observed on and around remnants of original tubules embedded in the gel. In noncontracted cultures there was no focal cell clustering and cells were squamous-like with rudimentary microvilli, similar in appearance to cells grown on plastic culture dishes. Measurements made in contracted monolayers yielded an average transepithelial resistance of 6.5 Ω cm², a spontaneous transepithelial potential difference of +0.9 mV, measured with respect to the serosa, and an apical membrane potential of −75 mV when cells were bathed in 0.4 mM K and −49 mV when cells were bathed in 4 mM K media. Mucosal protamine (50 μg/ml) increased transepithelial resistance by 22%, suggesting that the epithelial cell tight junctions were responsive to external stimuli. Monolayers were anion selective, giving a dilution potential (lumen-directed NaCI gradient) of −2.6 mV with respect to the serosa.

These experiments show that primary culture of rabbit renal cortical cells separated by differential sieves displays electrophysiologic and morphologic characteristics of a proximal renal tubular epithelium. Confluency and attainment of differentiated morphology and function are promoted when monolayer cells are not bound to an unyielding substrate.

Tumor necrosis factor (TNF) is a monokine which is involved in macrophage-mediated cytotoxicity (MMC). We have previously reported that peroxidases can activate thioglycollate-induced macrophages to the tumoricidal state in vitro. The present study was undertaken in an attempt to correlate peroxidase-induced MMC with production of TNF. Horseradish peroxidase (HRP) was used as the principal model for these studies. Resident and thioglycollate-induced macrophages exposed to peroxidases were examined for both MMC against 3T12 cells and production of TNF. Thioglycollate-induced macrophages exposed to HRP, bovine lactoperoxidase, or human myeloperoxidase demonstrated enhanced secretion of TNF. When exposed to HRP, both resident and thioglycollate-induced macrophages secreted significant amounts of TNF and acquired the ability to lyse 3T12 cells. However, resident macrophages were considerably less efficient in both their cytotoxic activity and TNF secretion. Macrophage-mediated cytotoxicity was eliminated by the addition of specific antisera to TNF. In addition, replacement of culture supernatants within 24 hr after exposure of the macrophages to HRP increased tumor cell killing in the absence of additional detectable TNF production, suggesting that other factors may be involved in peroxidase-induced MMC. These results indicate that TNF is intimately associated with peroxidase-induced MMC and suggest a possible role for peroxidases as immunomodulators via augmentation of macrophage capacities and functions.

Earlier studies have revealed an impairment of jejunal absorption of long chain fatty acids in experimental uremia. We investigated the intestinal absorption of butyric acid which is a short chain fatty acid in experimental renal failure (RF). Sprague-Dawley rats were randomized into the RF group which had subtotal nephrectomy, a sham-operated control group, and a pair-fed group. In vivo recirculating perfusion (n = 5) and in vitro everted sac incubation (n = 8) were employed. The in vitro experiments were repeated substituting the serosal buffer by either predialysis or postdialysis sera from uremic individuals, or normal serum (n = 10). The rate of in vivo butyric acid absorption was significantly lower while the in vitro absorption was significantly higher in the RF group than those observed in the sham-operated and pair-fed groups which showed comparable values. The normality of butyric acid absorption in the pair-fed animals despite comparable weight loss with the RF group tends to exclude anorexia and weight loss as a cause of altered butyric acid transport in RF animals. The disparity between the in vivo and in vitro data is suggestive of an inhibitory influence of uremic environment which is present in vivo and absent in vitro. This viewpoint was corroborated by the observed fall in butyric acid absorption by sacs containing predialysis uremic serum as compared with those containing normal or postdialysis sera. The latter further suggests that the inhibitory factor(s) is dialyzable.

In order to evaluate the role of the α-adrenergic system in the systemic and renal hemodynamic changes of the acute combined blood gas derangement, seven conscious mongrel dogs in careful sodium balance (80 mEq/day for 4 days) were evaluated. Each animal was evaluated during combined acute hypoxemia (PaO₂ = 35 ± 1 mm Hg) and hypercapnic acidosis (PaCO₂ = 56 ± 2 mm Hg; pH = 7.18 ± 0.01) with (i) vehicle (D5W) alone and (ii) α ₁-adrenergic blockade with prazosin, 0.1 mg/kg iv. Mean arterial pressure increased during the combined blood gas derangement with vehicle. In contrast, mean arterial pressure fell during combined acute hypoxemia and hypercapnic acidosis with α ₁-adrenergic blockade. The mechanism for abrogation of the rise in mean arterial pressure during the combined blood gas derangement by α ₁-adrenergic blockade appeared to be through attenuation of the rise in cardiac output rather than an exaggerated fall in total peripheral resistance. These observations suggest that the α-adrenergic system is important in circulatory homeostasis during the combined blood gas derangement.

Amiodarone, a class III antiarrhythmic drug, has been found to be effective in the management of patients with life-threatening ventricular arrhythmias. Recent reports describe the presence of myelinoid inclusion bodies following amiodarone therapy in liver, myocardium, white blood cells, lung, cornea, skin, and lymph nodes; their relationship to toxicity is unclear. The exact role of desethylamiodarone, the major metabolite, of amiodarone in systemic toxicity of the parent drug is not known. Concentration-response relationships for amiodarone and desethylamiodarone were investigated by adding 1–50 μg/ml of the compounds of dimethyl sulfoxide (controls) to hepatocytes isolated from Sprague-Dawley rats and cultured in Lelbovitz L-15 medium. Using lactate dehydrogenase release into the medium to quantitate cell death, both drugs were found to cause cell death in a concentration-dependent manner within 24 hr of incubation; this data showed desethylamiodarone to be significantly more toxic than amiodarone. In experiments with 50-μg/ml concentrations of amiodarone or desethylamiodarone, we found desethylamiodarone to produce a significantly greater release of lactate dehydrogenase as compared with amiodarone within 2–4 hr. Electron microscopic studies indicated the presence of myelinoid inclusion bodies at early culture stages followed by progressive swelling of mitochondria and rough endoplasmic reticula, disruption of membranes, aggregation of subcellular structures, and ultimately cell death. Ultrastructural changes occurred sooner in the hepatocytes treated with desethylamiodarone than with amiodarone. These data demonstrate that (i) desethylamiodarone is more toxic than amiodarone; (ii) acute toxicity of desethylamiodarone and amiodarone can be quantitated by lactate dehydrogenase release; (iii) both desethylamiodarone and amiodarone can induce myelinoid inclusion bodies in cultured hepatocytes; and (iv) toxicity is characterized by progressive subcellular changes leading to cell death.

γ-Glutamyl transpeptidase (γ-GT) localization in exfoliative esophageal specimens was evaluated in a population of 911 individuals aged 35 or older who lived in an area of China with a high incidence of esophageal squamous carcinoma. Of the total population, 24.4% was positive for γ-GT. Graded histopathologic cellular classification revealed the following incidences: normal, 10.4% (43 of 413); hyperplasia, 22.0% (44 of 200); Grade I dysplasia, 42.4% (98 of 231); Grade II dysplasia, 46.0% (23 of 50); near-carcinoma, 100% (4 of 4); and squamous carcinoma, 77.0% (10 of 13). In these groups γ-GT-positive cells were usually from normal esophageal squamous epithelium. A lower incidence of γ-GT-positive cells was found for dysplastic cells (dysplasia grade I, 6.5% (15 of 231); grade II, 10.0% (5 of 50). The presence of γ-GT-positive cells in normal esophageal squamous epithelium may prove useful for identification of suspect populations; the presence in dysplastic cells may serve as a diagnostic marker for patients who will soon progress to the carcinoma stage and who should receive early treatment and careful follow-up.

Plasma ¹²⁵I-albumin was used as a marker of extracellular dilution in order to study the effect of high-speed centrifugation on transmembrane water distribution in several types of human red cells, including normal (AA), hemoglobin variants (βA, AS, SC, βS, and SS), and those from patients with hereditary spherocytosis. SS and AA erythrocytes were also examined for changes in intracellular hemoglobin concentration of three different density fractions and with increasing duration of spin. The minimum force and duration of centrifugation required to impair water permeability were found to vary with the red cell type, the anticoagulant used (heparin or EDTA), the initial hematocrit of the sample centrifuged, as well as among the individual erythrocyte fractions within the same sample. When subjecting pathologic erythrocytes to high-speed centrifugation, the ¹²⁵I-albumin dilution technique can be used to determine whether the centrifugation procedure has led to an artifactual red cell water loss and to correct for this when it does occur. An abnormal membrane susceptibility to mechanical stress was demonstrated in erythrocytes from patients with hereditary spherocytosis and several hemoglobinopathies.

The blood pressure-lowering potency and activity of BRL 34915, a new vasodilator and putative stimulator of potassium efflux from vascular smooth muscle, was investigated in conscious spontaneously hypertensive rats (SHR) and normotensive rats (NTR) after intravenous administration and compared with that of the calcium channel blocker, nifedipine. In SHR, BRL 34915 (3–100 μg/kg) or nifedipine (10–3000 μg/kg) produced similar reductions in mean arterial pressure of 58 ± 3% and 55 ± 3%, respectively. BRL 34915 (ED_30% = 13.8 μg/kg) was 15.3 times more potent than nifedipine (ED_30% = 207 μg/kg) in SHR. In contrast, only a 1.7-fold difference in potency was observed in NTR between BRL 34915 (ED_30% = 123 μg/kg) and nifedipine (ED_30% = 182 μg/kg). The potency ratio (ED_30% NTR/ED_30% SHR) for BRL 34915 was 8.83 whereas nifedipine had a ratio of 0.88, reflecting the greater responsiveness of the SHR to BRL 34915. Systemic hemodynamics were monitored in anesthetized SHR and NTR to determine the basis for the reductions in blood pressure. BRL 34915 (3–100 μg/kg iv) lowered mean arterial pressure in both groups solely by decreasing total peripheral vascular resistance, since no changes in cardiac output were observed. Relaxation responses were also obtained in phenylephrine-contracted isolated aortic strips from both strains of rat to ascertain whether differences in responsiveness existed at this level of the vasculature. No significant difference in the potency of BRL 34915 (3–10 μM) as a vasodilator was found in aortas from SHR or NTR. These results indicate that, unlike nifedipine, BRL 34915 is a more potent vasodepressor agent in SHR than in NTR and suggests that the potassium efflux stimulator may preferentially relax resistance vessels in the hypertensive rat.

The presence of type II 5′-deiodinase activity in the Syrian hamster Harderian gland was investigated. This enzyme exhibited an increase of its activity after animals entered the normal dark phase, with maximal activity occurring at 04.00 hr (8 hr after lights off). The nocturnal increase was prevented by maintaining the animals in light during the night. Isoproterenol subcutaneously injected every 2 hr (1.0 mg/kg body wt) from 20.00 hr through 0.400 hr to animals exposed to light during the normal dark period mimicked the effect of darkness, i.e., with this treatment an increase in 5′-deiodinase activity with maximal peak values at 02.00 hr was observed. The results show that 5′-deiodinase activity in the Syrian hamster Harderian gland exhibits a nyctohemeral profile dependent on β-adrenergic activation of the gland.

Pineal thyroxine 5′-deiodinase (5′-D) activity rose > 10-fold above the basal level 2–3 hr after 1 mg/kg isoproterenol and returned to near the basal level by 6 hr. The same dose of norepinephrine or phenylephrine was without effect, but phenylephrine modestly potentiated isoproterenol-stimulated 5′-D activity. Either actinomycin D or cycloheximide treatment markedly decreased diurnal isoproterenol stimulation and the spontaneous rhythmic nocturnal rise of pineal 5′-D. The data indicate that pineal 5′-D activity is very similar to pineal serotonin N-acetyl transferase in being primarily stimulated by β-adrenergic agonists and requiring new RNA and protein synthesis for its activation.

The influence of major histocompatibility complex (B complex) dosage on monocyte-macrophage function was examined using 4- to 6-week-old trisomic strain chickens. Di- (B¹⁵ B¹⁵ ), tri- (B¹⁵ B¹⁵B¹⁵ ), and tetrasomic (B¹⁵B¹⁵ B¹⁵B¹⁵ ) progeny were produced from trisomic x trisomic crosses. Although mononuclear leukocytes from tetrasomics exhibited enhanced chemotactic activity in response to both f-met-leu-phe and Enterobacter cloacae culture supernatant as compared with that of cells from other groups, the ability to generate peritoneal exudate cells in response to intraperitoneal Sephadex stimulation was similar in all groups. Among peritoneal exudate cells, tetrasomic birds produced a significantly lower percentage of adherent macrophages with a higher proportion of Fc receptor-positive and CMTD-2-reactive macrophages than either disomic or trisomic chickens. Both tetrasomic and trisomic peritoneal macrophages exhibited a reduced phagocytic activity for unopsonized but not opsonized SRBC than was found with disomic macrophages. Thus, the number of major histocompatibility complex copies present in cells appears to influence monocyte-macrophage function.

In vitro actions of purified plerocercoid growth factor (PGF) were compared with those of insulin and human growth hormone (hGH) in adipose tissue from normal male rats. Insulin-like effects were measured by the ability of PGF, insulin, or hGH to stimulate oxidation of [U-¹⁴C]glucose to ¹⁴CO₂, to stimulate lipogenesis, and to inhibit epinephrine-induced lipolysis. PGF and insulin stimulated significant increases in glucose oxidation and lipogenesis in adipose tissue that had not been preincubated as well as in tissue that had been preincubated. hGH stimulated insulin-like effects only in tissue that had been preincubated for 3 hr. Insulin, hGH, and PGF inhibited epinephrine-induced lipolysis of preincubated (3 hr) adipose tissue. hGH produced a dramatic lipolytic response in tissue freshly removed from normal rats but no dose of PGF was lipolytic. PGF did not displace ¹²⁵I-insulin from its receptors on adipocytes but did competively inhibit ¹²⁵I-hGH binding to adipocytes. These results suggest that PGF has direct insulin-like actions which are initiated by binding a GH receptor, but PGF had no anti-insulin action and the insulin-like activity of PGF was unaffected by refractoriness of adipose tissue to GH.

Rats were fed diets containing either soy protein or casein and different levels of manganese, methionine, phytic acid, or arginine for 7 days and then fed test meals labeled with 2 μCi of ⁵⁴Mn after an overnight fast. Retention of ⁵⁴Mn in each rat was measured every other day for 21 days using a whole-body counter. Liver manganese was higher (P < 0.0001) in soy protein-fed rats (8.8 μg/g) than in casein-fed rats (5.2 μg/g); manganese superoxide dismutase activity also was higher in soy protein-fed rats than in casein-fed rats (P < 0.01). There was a significant interaction between manganese and protein which affected manganese absorption and biologic half-life of ⁵⁴Mn. In a second experiment, rats fed soy protein-test meals retained more ⁵⁴Mn (P < 0.001) than casein-fed rats. Liver manganese (8.3 μ/g) in the soy protein group was also higher than that (5.7 μg/g) in the casein group (P < 0.0001), but manganese superoxide dismutase activity was unaffected by protein. Supplementation with methionine increased ⁵⁴Mn retention from both soy and casein diets (P < 0.06); activity of manganese superoxide dismutase increased (P < 0.05) but liver manganese did not change. The addition of arginine to casein diets had little effect on manganese bioavailability. Phytic acid affected neither manganese absorption nor biologic half-life in two experiments, but it depressed liver manganese in one experiment. These results suggest that neither arginine nor phytic acid was the component in soy protein which made manganese more available from soy protein diets than casein diets.

A modification of an existing separation technique by this laboratory is described for the separation and quantification of the three commercially available forms of ascorbic acid. The technique has the potential for identifying the various metabolic and degradation products resulting from vitamins C2 and C3 metabolism. A microwave technique is used for tissue heat denaturation and extraction.