Experimental Biology and Medicine

The ability of ethanol to reduce α-adrenergic receptor-mediated pressor responsiveness in vivo was investigated in chloralose-anesthetized male Sprague-Dawley rats. Catheters were inserted in the jugular vein and the femoral artery of rats for the injection of drugs and the measurement of blood pressure, respectively. Dose-response curves for phenylephrine and norepinephrine were constructed by plotting the change in mean arterial pressure following a bolus dose of the agent against the dose of the pressor agent used. Following construction of an initial dose-response curve, animals were challenged with either a 1 g/kg dose of ethanol or an equivalent volume of saline (iv) and the dose-response curves were repeated. Using a similar protocol, pressor responsiveness was evaluated in animals pretreated with either yohimbine (1 mg/kg) or prazosin (3.9 μg/kg), a dose sufficient to produce partial blockade of α receptor-mediated pressor responsiveness, and then treated with ethanol. Ethanol produced a partial blockade of α receptors when the animals were challenged with either phenylephrine or norepinephrine. This blockade produced by ethanol was shown to be similar to that produced by the receptor blocking agents used in this study. To rule out any nonspecific effects of ethanol in reducing vascular reactivity, some animals were challenged with angiotensin II both before and after treatment with ethanol, yohimbine, or prazosin and after both drugs were administered together. Ethanol, as well as the α,₁- and α ₂-adrenergic blocking agents tested failed to have any significant effect on angiotensin II-pressor responsiveness, ruling out any nonspecific effect of ethanol on the vasculature. It is concluded, therefore, that ethanol has α receptor blocking-like activity in vivo.

Norcocaine (NC) and N-hydroxynorcocaine (NHNC), products of the oxidative metabolism of cocaine, were examined in plasma, brain, and liver of mice injected intraperitoneally with cocaine. Plasma levels of NHNC were altered in vivo by inhibiting esterase activity with diazinon and chloral hydrate or activating esterase activity with phenobarbital, and activating the microsomal P-450 system with phenobarbital. Changes in plasma concentrations of NHNC resulted in similar changes in brain, which were often different from those in liver. After intracisternal administration of cocaine to mice, no appreciable amount of NC or NHNC could be detected in brain; the same results were obtained upon intracisternal and intraventricular administration to rats. Microsomal preparations from mouse brain were found to be considerably less active than those from liver in converting NC to NHNC. We conclude that the cerebral oxidative metabolism of cocaine is not appreciable and that most of the NC and NHNC found in the brain after systemic cocaine administration is derived from plasma rather than formed centrally by brain microsomal enzymes.

This study was conducted to determine whether progesterone inhibits luteinizing hormone (LH) secretion in female pigs by a direct action on the pituitary gland. Eight ovariectomized, hypophysial stalk-transected gilts were given 1-μg pulses of gonadotropin-releasing hormone iv every 45 min from Day 0 to 12. On Days 5–12, each of four gilts received either progesterone or oil vehicle im at 12-hr intervals. Serum progesterone concentrations in steroid-treated gilts reached 70 ± 6.8 ng/ml (mean ± SE) by Day 8 and remained elevated thereafter, whereas serum progesterone concentrations in oil-treated controls were less than 1 ng/ml for the entire study. Daily serum LH concentrations were not different between gilts treated with progesterone or oil. The 1-μg pulses of gonadotropin-releasing hormone reliably evoked pulses of LH in both treatment groups. The LH pulse frequency and amplitude, assessed from samples collected every 15 min for 6 hr on Day 12, were similar for progesterone- and oil-treated gilts. These results provide evidence that progesterone does not act at the pituitary gland to alter LH secretion in pigs.

Intracerebroventricular administration of either rat atrial natriuretic factor (99–126) or dopamine to conscious male hydrated rats resulted in an increase in urinaryvolume and sodium excretion. This activity was prevented, in both cases, by nonselective dopamine antagonist haloperidol (2.5 or 1.25 mg/kg sc, 18 and 2 hr before intracerebroventricular administration of atrial natriuretic factor). Our findings suggest that atrial natriuretic factor exerts its centrally mediated effects on sodium and water metabolism, at least in part, via a dopaminergic mechanism.

Rhodamine B-labeled synthetic latex particles (microspheres), 1.8 μm in diameter, were administered by gavage 5 days per week to young (24 days) and aged (18 months) mice. After 25 days (19 gavages), the particles were assayed in solubilized tissues by depositing them on filters and counting under fluorescence microscopy. Aged mice exhibited significantly more fluorescent particle accumulation in Peyer's patches but significantly less in lungs than young mice. Mesenteric lymph nodes and Peyer's patch–free intestinal segments contained measurable latex, but differences between young and aged animals were not significant. Liver contained only trace amounts of latex, and spleen and kidney were latex free in both young and aged animals. Nonquantitative observations on KOH-glycerol-cleared whole Peyer's patches and slices of liver, lung, and mesenteric lymph node were similar.

Calcium-containing crystals and elevated levels of calcium chloride (CaCl₂) and lanthanum chloride (LaCl₃) have been previously reported to enhance the proliferative activity of cultured fibroblasts. We have investigated the relative mitogenicity of these agents, whether they function via precipitation on the cell surface and whether they interact with one another. Confluent cultures of newborn foreskin fibroblasts provided with fresh medium containing 10% fetal bovine serum (FBS) in the presence of hydroxyapatite (HA), pyrophosphate (PP_i), LaCl₃ (La), or additional CaCl₂ (Ca) were all stimulated more than control cultures provided with fresh medium and 10% FBS alone as assessed by cell counts 5 days later. Increases in cell yield above the original confluent cell density were 316% for La, 271% for Ca, 189% for HA, 131% for PP_i, and 45% for controls. Addition of fresh medium containing 10% FBS and epidermal growth factor or fresh medium containing 20% FBS as additional points of reference yielded increases of 204 and 107%, respectively, over original confluent density. Stimulation induced by La or Ca was significantly greater (P < 0.001) than the stimulations induced by each of the other treatments. The same treatments added to confluent cultures without a change of medium also renewed mitotic activity, with La and Ca again the most mitogenic and approximately doubling the pretreatment cell yields. Cultures incubated in an inverted position to avoid cell contact with precipitates in the medium were also stimulated by La and Ca, but not by HA and PP_i. When added to confluent cultures simultaneously supplemented with optimal additional Ca, La decreased Day 5 cell yields in a dose-dependent manner at low concentrations (0.03–0.2 mM) but increased cell yields over those obtained with 0.2 mM LaCl₃ again in a dose-dependent manner at higher concentrations. Thus, while HA and PP_i act via precipitation on the cell surface, the more mitogenic agents La and Ca function in solution and appear to stimulate cell division by different nonadditive mechanisms. These findings suggest multiple mechanisms of membrane participation in mitogen responsiveness and in density-dependent inhibition of growth.

Vanadate is known to have an insulin-like action which stimulates sugar transport in some systems like adipocytes and muscle cells, but in other systems it inhibits sugar transport by decreasing the activity of (Na⁺ +K⁺)-ATPase. To evaluate whether these two opposing actions may influence sugar transport across the intestine, we studied the effects of acute and chronic vanadate administration on the uptake of glucose, galactose, and 3-O-methylglucose in isolated rat intestinal cells. The sugar uptake measurements were also coupled by determinations of rubidium-86 uptake as a measure of the activity of the Na-K pump. Both acute and chronic vanadate administration reduced rubidium uptake by the cells but the reduction did not uniformly influence the uptakes of the three sugars in question which were stimulated by the acute exposure of the cells to vanadate. Glucose uptake was also stimulated by chronic vanadate administration, but the uptakes of galactose and 3-O-methylglucose were respectively unaffected or inhibited by chronic vanadate. The findings suggest that the effect of vanadate on sugar transport is dependent on the net difference between two actions of vanadate: (i) stimulation of a receptor site (possibly an insulin receptor site) in the intestinal cell membrane and (ii) inhibition of the Na-K pump. During acute vanadate exposure, the stimulation of the receptor site was very likely a dominant feature which overwhelms the inhibition of the pump. Chronic exposure to vanadate led, on the other hand, to only a limited degree of stimulation of the receptor site and the inhibition of the Na-K pump became evident in the uptake measurements of galactose and 3-O-methylglucose. Glucose uptake, however, was stimulated by chronic vanadate ingestion due, very likely, to an increase in the metabolism of this sugar which occurred only with prolonged exposure of the rat intestine to vanadate.

An initial study was conducted to establish the presence in plasma of diurnal rhythms of immunoreactive porcine adrenocorticotropic hormone (pACTH) and cortisol in castrated male pigs (barrows). Fourteen barrows with jugular catheters were bled at 6-hr intervals for 24 hr. Significant changes in plasma pACTH were evident with peak levels (61 ± 6 pg/ml) at 0100–0700 hr and a trough (38 ± 4 pg/ml) at 1900 hr. Changes (P < 0.05) in plasma cortisol were also present in barrows with a peak (44 ± 6 ng/ml) at 0700 hr and a trough (21 ± 5 ng/ml) at 1900 hr. Plasma norepinephrine and epinephrine were measured at the same time intervals and did not differ among hours. In these unstressed pigs the ratio cortisol/log₁₀ pACTH at 0700 hr (25.3 ± 3.0) was greater than the ratio at 1900 hr (12.9 ± 2.7). Sequential blood samples were subsequently taken on four of the barrows 12 and 26 days later. Plasma pACTH was variable among pigs and did not differ among hours. Plasma cortisol on both dates was greater (P < 0.05) in the morning (0100 or 0700 hr) than at 1900 hr. The ratio cortisol/log₁₀ pACTH at 0700 hr was repeatedly greater than at 1900 hr. A second study was conducted to determine whether plasma pACTH and cortisol responses to mild (32°C for 2 hr) or strong (20-min restraint) stressors were dependent on the time of day of stressor application (0800 hr, AM; 1600 hr, PM). Response-associated parameters (maximum concentration, maximum incremental concentration, and integrated response) for pACTH and cortisol did not differ between AM and PM. However, a qualitative difference existed between the AM and PM plasma pACTH responses to restraint +32°C wherein the AM response consisted of a single prolonged surge, and the PM response of an initial major peak followed by a second significant minor peak. A suggested explanation is that the initial 20-min restraint stressor potentiated the hypothalamic-hypophyseal response to 32°C. These studies are the first direct measurements which suggest the presence of diurnal changes in plasma ACTH and cortisol in barrows. The studies also indicate for barrows an absence of diurnal changes in plasma epinephrine and norepinephrine. The responsiveness of the pituitary-adrenocortical axis to stressors did not exhibit quantitative diurnal changes at the time periods measured. However, it is hypothesized that the repeatable AM-PM difference in the ratio cortisol/log₁₀ ACTH reflects a diurnal change in adrenal responsiveness to ACTH in unstressed pigs.

Both human lysozyme (HL) and hen egg white lysozyme (HEWL) inhibited the proliferative response of peripheral blood lymphocytes to T cell mitogens such as the lectins phytohemagglutinin and concanavalin A. This inhibition was observed both when HL or HEWL was added to the lymphocyte cultures in combination with phytohemagglutinin or concanavalin A and when lymphocytes were pretreated with either lysozyme and extensively washed prior to culture with mitogens. Under both conditions, the effects were strictly dose dependent; the lysozyme concentrations yielding maximal inhibitory effect were 5 μg/ml for HL and 1 μg/ml for HEWL, while both lower and higher concentrations were less effective. Specific antilysozyme rabbit sera completely prevented the inhibitory effects of both HL and HEWL on the proliferative response of lymphocytes to phytohemagglutin or concanavalin A. Chitotriose (a lysozyme inhibitor) caused a strong reduction in the inhibitory effects of the two lysozymes on the lymphocyte response to either lectin. HL and HEWL also were found to markedly inhibit the polyclonal B cell proliferation and differentiation induced by pokeweed mitogen and T cells. A less marked inhibition was also obtained when T cells, but not B cells, were pretreated with HL or HEWL. Again, as in the experiments with T cell mitogens, the effects were dose dependent and 5 μg/ml HL and 1 μg/ml HEWL proved to be the most effective concentrations. The possible mechanisms by which lysozyme inhibits the lymphocyte response to mitogenic lectins are considered and discussed. The enzymatic activity seemed to perform an essential function, as shown by the loss of effect when the heat- or trypsin-inactivated lysozymes were used and by the fact that only the enzymatically active compound, among certain semisynthetic derivatives of HEWL, inhibited the lymphocyte response to the mitogens. However, the cationic properties of the lysozyme molecule appeared to be essential too, since enzymes with a similar specificity of action showed effects similar to those observed with HL or HEWL only when they carried a strong positive charge. It is suggested that lysozyme, which is naturally secreted by monocytes and macrophages, might interact with lymphocyte surface receptor sites and participate in the complex mononuclear phagocyte-lymphocyte interactions and in the modulation of lymphocyte activation.

The present study was designed to measure the relationships between maternal arterial glucose concentration ([GI]_A) and fetal arterial glucose concentration ([GI]_a), uteroplacental glucose consumption (UPGC), and the rate of uteroplacental glucose transfer to the fetus (UPGT) in pregnant sheep in late gestation. [GI]_A was controlled by a glucose clamp technique and the glucose flux rates of the uteroplacenta were quantified by the Fick principle. [GI]_A varied from 1.81 to 154.7 mg/dl; [GI]_a was directly related to [GI]_A: [GI]_a = 0.374 [GI]_A + 1.81, r = 0.873, P < 0.001. Fetal arterial blood oxygen content decreased with [GI]_A (P < 0.05) and fetal arterial blood lactate concentration increased with [GI]_A (P < 0.001). There was no significant effect of [GI]_A on the rates of uteroplacental lactate production, uteroplacental oxygen consumption, fetal oxygen consumption, or uterine or umbilical blood flow. Both UPGC and UPGT were directly related to [GI]_A: UPGC = −2.221 × 10^-3 χ ² + 0.646 × −6.016, r = 0.80; UPGT = −1.208 × 10^-3 χ ² + 0.405 × −2.416, r = 0.90. UPGC and UPGT were approximately parallel over the range of [GI]_A studied (UPGC = 1.19 UPGT + 3.79, r = 0.764). These results demonstrate the importance of UPGC to maternal-fetal glucose homeostasis and indicate that factors regulating uteroplacental glucose consumption and transfer to the fetus become limiting at comparable levels of [GI]_A and [GI]_a. The estimated kinetic constants for UPGC represent the metabolism of glucose by the uteroplacental tissues, but the estimated kinetic constants for UPGT represent the metabolism of glucose by the fetus as well as the transfer of glucose by the uteroplacenta to the fetus.

The LD₅₀ for encephalitis caused by Semliki forest virus in 6– to 8-week-old mice is 1 plaque-forming unit (PFU) in C3H/Ten strain of mice when injected intracerebrally, iv, or in the footpad; however, the LD₅₀ by the ip route is 4 × 10³ PFU. In the ICR strain of mice at the same age, the LD₅₀ for the intracerebral route is 1 PFU, 10³ PFU for the iv and footpad routes, and 4 × 10³ PFU for the ip route. A number of in vivo and in vitro experiments were done to explain the relative resistance to Semliki forest virus injection by the ip route. The results suggest that the viruses are adsorbed to and enter adherent cells of the peritoneal cavity but do not replicate and release progeny virus. After inoculation with the virus, viral antigens could only be observed in methanol-treated cells as a halo by immunofluorescence at or just below the plasma membrane of only a small fraction (<0.5%) of peritoneal adherent cells. Naturally occurring interferon-α/β (<1 unit/ml) was found to probably play a marginal role, if any, in the resistance.

An anti-inflammatory factor isolated from milk of hyperimmunized cows was analyzed in vitro and in vivo. Macrophages collected from lacteal secretions of a unimmunized nonlactating cow showed increased ability to kill phagocytosed Staphylococcus aureus when incubated with the anti-inflammatory factor. Mice injected intraperitoneally with 10 mg/kg of anti-inflammatory factor demonstrated an increased LD₅₀ to S. aureus when challenged intraperitoneally. Injected mice also demonstrated significantly (P < 0.05) less mammary inflammation and involution and increased clearance of S. aureus when challenged intramammarily. Quantitative histologic analysis of mammary tissues from mice injected with antiinflammatory factor demonstrated significantly (P < 0.05) more lumen, less interalveolar connective tissue, and less leukocytic infiltration compared with control mice. Mammary glands of mice injected with anti-inflammatory factor and challenged with S. aureus also contained fewer colony-forming units than control mice. The product appeared to exert its effect on the nonspecific defense system via modulation of leukocyte function.

Previous studies have shown that administration of synthetic atrial natriuretic factor (ANF, 101–126) decreases sodium-dependent phosphate transport across renal brush border membrane vesicles (BBMV) in rats fed a normal or low phosphate diet. In the present study, infusion of rat ANF (atriopeptin III (ANP-III), 103–126 rat ANF) to rats fed a normal phosphate diet caused natriuretic and phosphaturic effects similar to those of ANF (101–126), but unlike ANF (101–126) did not increase the glomerular filtration rate. The effect of ANP-III infusion on sodium-dependent transport of phosphate was also determined in BBM vesicles isolated from the superficial cortex (BBMV-SC) and juxtamedullary cortex (BBMV-JM). The results indicate that ANP-III decreases phosphate transport across BBMV-SC and BBMV-JM similarly (20–24%). However, it had no effect on sodium-dependent transport of proline in these vesicles.

The infusion of ANP-III to rats fed a normal phosphate diet inhibits phosphate uptake both in BBMV-SC and BBMV-JM and causes phosphaturia without increments in glomerular filtration rate.

Rabbit antisera to rat pituitary follicle-stimulating hormone (FSH) and to rat luteinizing hormone (LH) were used, in an immunocytochemical probe, to determine the ontogeny and distribution of immunoreactive, endogenous, intraovarian FSH and LH in immature rats. Ovaries from rats 4, 8, 12, and 21 days of age were studied. Both gonadotrophins were first immunodetectable on Day 8. In reactive primordial follicles, LH was restricted to the cytoplasm and nuclei of the surrounding follicle cells. In those follicles possessing both squamous and cuboidal follicle cells, i.e., transitional between primordial and primary, LH was found in both the cytoplasm and nuclei of both follicle cell types. In primary follicles, LH was no longer present in granulosa cells but was concentrated in germ cell cytoplasm. In contrast, in primordial follicles, FSH was restricted to the germ cell but was present in both the oocyte cytoplasm and germinal vesicle. In transitional and primary follicles, FSH remained within the oocyte cytoplasm and germinal vesicle but also became detectable within the cytoplasm and nuclei of granulosa cells. These findings raise some important new questions regarding the role(s) of the gonadotrophins in early follicular development.

Copper deficiency adversely affects the extracellular matrix of the arterial wall, leading to cardiovascular lesions. To study the lesions resulting from copper deficiency, the composition of proteoglycans from aortas of copper-deficient rats was compared with proteoglycans of aortas from copper-supplemented rats. Copper deficiency in rats was verified by copper levels in adrenal glands (mean ± SE, 0.37 ± 0.07 vs 1.03 ± 0.17 μg/g wet wt in supplemented rats). The proteoglycans were isolated from the aorta by extraction with 4 M guanidine-HCl and by digestion of the tissue with elastase. The proteoglycans were purified by CsCl isopycnic centrifugation and fractionated by gel filtration. The fractions were characterized for molecular size and glycosaminoglycan composition. Total uronate in the aortas from copper-deficient rats was 25% greater than in aortas from copper-supplemented rats, and the proteoglycans from copper-deficient rat aortas were of greater molecular size. Among the glycosaminoglycans the concentration (μg/mg tissue) of isomeric chondroitin sulfates, particularly dermatan sulfate, was greater in copper-deficient animals than in copper-supplemented animals. These observations are similar to earlier findings in experimental atherosclerosis and to a response of cardiovascular connective tissue to injury.

Young adult male rats were treated with isoproterenol during the day to induce high levels of pineal N-acetyltransferase (NAT) activity and melatonin. Roughly 2 hr later when pineal NAT activity and melatonin levels were elevated, animals were given either an injection of a calcium channel blocker, i.e., either nifedipine or verapamil, or diluent. The rats were then forced to swim for 10 min in room temperature (22°C) water. Fifteen minutes after swimming onset, pineal glands were collected for measurement of NAT activity and melatonin. Swimming caused a dramatic reduction in pineal melatonin content without influencing NAT activity. Nifedipine substantially and verapamil completely blocked the drop in pineal melatonin levels due to swimming without influencing NAT activity. The results suggest that calcium may be somehow directly or indirectly involved in melatonin release from the rat pineal gland.

Lithium (Li) is a known agent capable of producing leukocytosis, first observed in manic depressive patients receiving Li as therapy; however, a certain percentage of cases are nonresponsive to Li therapy. These patients are responsive to carbamazepine (CBZ); however, severe hematopoietic side effects have been associated with CBZ treatment such as leukopenia. We report here the results of dose-response studies (0.1–100 μg/ml) that demonstrate CBZ treatment inhibits both murine and human bone marrow-derived granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and megakaryocyte progenitor (CFU-Meg) cells. The addition of Li prior to and simultaneously with CBZ to marrow cultures was effective in reversing the CBZ-induced toxicity only in the presence of an optimal Li dose (1.0 mM) known to stimulate bone marrow function. However, when the addition of Li was delayed 24 hr to CBZ-treated cultures no protective effect was observed for any marrow progenitor. Thus, the time of Li-CBZ exposure is critical to observe the protective effect of Li. These results demonstrate that the leukopenia associated with CBZ treatment may be due to the abiity of CBZ to inhibit marrow progenitor cells and suggest Li may be an effective agent to reverse the marrow toxic effects of CBZ.

It is often necessary to have a small animal model which permits the sequential evaluation of functional immune status over a period of time. We report here the in vivo, intradermal response to phytohemagglutinin which produces an area of induration that is histologically similar to a typical delayed cutaneous hypersensitivity response, and that provides fast, quantitative, reproducible results similar to those observed with standard but more laborious and variable in vitro tests of immune function. For small animal studies this has the advantage of permitting longitudinal evaluations over time without sacrificing the animal. Using phytohemagglutinin-microprotein (0.2 mg/0.1 ml), injected intradermally, a delayed cutaneous hypersensitivity-like response is induced which is maximal at 24 hr. When immune function was altered either by treatment with a chemical immunosuppressant (ethanol) or by hormonal manipulations (hypophysectomy and rat growth hormone), the delayed cutaneous hypersensitivity-like response (area of induration) correlated closely with both macrophage migration inhibitory factor changes (r = 0.98; P < 0.001) and mixed lymphocyte reaction changes (r = 0.99; P < 0.05). These observations suggest that this technique correlates well with standard in vitro measures of immune response and may thus permit an in vivo estimation of immune reactivity.

Published reports indicate that Cu deficiency reduces antioxidant defenses and may result in tissue peroxidation. Dimethyl sulfoxide (DMSO), a highly penetrant antiinflammatory agent and purported hydroxyl radical scavenger, was chronically fed to male, weanling Sprague-Dawley rats which were either supplemented with or deficient in Cu. DMSO was found to inhibit the cardiac hypertrophy, anemia and depression of heart Cu concentration which occurs with Cu deficiency. This suggests that the hydroxyl free radical may contribute to the cardiovascular defects caused by dietary Cu deficiency.