Experimental Biology and Medicine

Noradrenergic mechanisms have a stimulatory role in regulating prolactin (PRL) secretion in the rat. We investigated the mechanism by which the α ₂-adrenergic system stimulates PRL release in urethane-anesthetized male rats. Intracerebroventricular injection of norepinephrine (2 μg/rat) or epinephrine (100 ng and 1 μg/rat) caused an increase in plasma PRL levels. The PRL increase induced by epinephrine was much greater than that by norepinephrine. Intracerebroventricular injection of phentolamine (1 μg/rat), an α-antagonist, blunted the plasma PRL increase induced by epinephrine (100 ng intracerebroventricularly). Plasma PRL levels were increased by intravenous injection of α ₂-agonists, clonidine (15 μg/100 g of body wt), and xylazine (200 μg/100 g of body wt). Plasma PRL increase induced by clonidine or xylazine was suppressed by intravenous injection of naloxone (125 μg/100 g of body wt), an opiate antagonist. These findings suggest that α ₂-adrenergic mechanisms stimulate pituitary PRL secretion, at least partly, by activating endogenous opioid peptides in the rat.

The cationic fluorescent probe, DiSC₃(5) was used to measure the membrane potential in human platelets. Hyperpolarization was induced by the addition of Ca²⁺ to the medium and also by the addition of the Ca²⁺ ionophore, A23187. In the absence of extracellular Ca²⁺ ([Ca²⁺]_o) there was no response to A23187. The threshold concentration for [Ca²⁺]_o was 20 μM and for A23187 was 12 nM. The increase polarity induced by [Ca²⁺]_o was not affected by various K⁺ channel blockers. However, the effect of A23187 was inhibited by quinine and charybdotoxin, while apamin, tetraethylammonium, and the calmodulin inhibitors trifluoperazine and compound R24571 were ineffective. The resting membrane potential was −66 ± 0.9 mV and was decreased by quinine. There are three conclusions from this study: (i) Ca²⁺-activated K⁺ channels exist in human platelets; (ii) they are the type that are apamin insensitive, charybdotoxin sensitive; and (iii) they may contribute to the resting membrane potential.

Exposure to hypobaric hypoxia (H: simulated altitude = 3658 m) was initiated in 5-week-old, male spontaneously hypertensive (SHR) and Wistar-Kyoto (WKy) normotensive rats while normoxic controls (N) for both groups were maintained under laboratory conditions. Significant attenuation of systolic arterial blood pressure was evident in SHR-H relative to SHR-N (125 ± 6 vs 145 ± 5 mm Hg; P < 0.05) but not in WKy-H relative to WKy-N (WKy-H, 116 ± 2 vs WKy-N, 117 ± 5 mm Hg). Hypoxia significantly decreased metabolic efficiency in both normotensive and hypertensive rats, although being both more severe and accompanied by significantly impaired growth rate in SHR-H. Urinary excretion of norepinephrine in the SHR was elevated relative to WKy, irrespective of altitude treatment, while hypoxia elicited similar increases in urinary excretion of norepinephrine in both SHR and WKy. Myocardial and adrenal contents of norepinephrine were significantly reduced following 3 days of simulated altitude exposure in both strains of rats. Tissue contents of norepinephrine in hypoxic rats returned to normoxic levels by 21 days of simulated altitude. Both urine and tissue indices provided consistent indirect evidence that changes in sympathetic neuronal activity in response to hypoxia were similar in normotensive and hypertensive rats. These findings suggest that prior reports of reduced α-adrenergic responsiveness in vasculature from hypoxia-exposed SHR reflect a postsynaptic event that is regulated independently of norepinephrine release from sympathetic nerve terminals.

The relative levels of elastin-specific mRNA were used as a measure of tropoelastin expression in uteri from pregnant Sprague-Dawley rats. The levels of elastin-specific mRNA were also correlated with values for net tropoelastin production and net deposition of mature, crosslinked elastin. The total content of uterine elastin increased throughout gestation, reaching maximal levels at Day 19 of gestation, which were three times those of nongravid tissue. Following involution, the elastin content decreased rapidly to near baseline values by 5 days postpartum. The content of soluble elastin, estimated using an enzyme-linked immunosorbent assay, paralleled in part the increase in elastin deposition and elastin mRNA levels. Uterine elastin metabolism appears to be unlike that in other elastic tissues, e.g., lung and large blood vessels. In most elastin containing tissues, the protein is synthesized during discrete developmental periods and is not readily degraded. However, uterine elastin is continuously expressed, and appears to be in a continual cycle of degradation and replacement.

The effects of thyroid manipulation on growth, feed efficiency, and plasma hormone levels were determined in rapidly growing chickens. Beginning at 3 weeks of age, eight broiler cockerels were provided with control feed (CF) or feed containing either 1 ppm of triiodothyronine (T₃), 1 ppm of thyroxine (T₄), 0.3% propylthiouracil (PTU), or 5 ppm of thyrotropin-releasing hormone (TRH) for 3 weeks. Blood samples were taken at 4, 5, and 6 weeks for determination of plasma levels of growth hormone, insulin-like growth factor, T₃, T₄, insulin, glucagon, glucose, and nonesterified fatty acids. Dietary TRH increased (P < 0.05) the growth rate of chickens by 14% when compared with the CF group. Plasma growth hormone levels were reduced (P < 0.05) 65% by dietary T₃ and 33% by treatment with either T₄ or TRH when compared with the CF group. Plasma insulin-like growth factor levels were 16% lower (P < 0.05) in PTU-fed birds than the other treatment groups. Plasma T₃ levels were elevated (P < 0.05) 3-fold by dietary T₃ and 38% by TRH whereas plasma T₃ in the PTU group was 38% below the average of CF birds. Plasma T₄ levels were increased (P < 0.05) by 12-fold in T₄-fed birds, decreased 48% in TRH-fed birds, and nondetectable in birds treated with either T₃ or PTU. Compared with the other treatments, dietary PTU increased (P < 0.01) plasma insulin levels 4.3-fold whereas TRH provided a 2.7-fold increase in plasma insulin. Plasma glucagon levels were 26% higher (P < 0.05) in T₃-fed birds than those fed either T₄ or PTU. These observations indicate that thyroid activity plays an important role in regulating secretion of GH and the pancreatic hormones. Furthermore, our study demonstrates the potential use of TRH as an orally active growth promoter for poultry.

Mammary tissue from five midpregnant heifers was transplanted subcutaneously into ovariectomized athymic mice (eight pieces/mouse). After a recovery period of 19 days, mice were injected daily for 5 days with buffer (50 mM NH₄HCO₃, pH 7.8) as control, 17β-estradiol (1 μg) plus progesterone (1 mg). Concurrently with the buffer or steroid hormone injections, mice were injected with bovine placental lactogen (0, 5, or 25 μg), bovine prolactin (0, 3.4, or 17.2 μg), or bovine growth hormone (0, 3.4, or 17.2 μg). All mice were injected with 2-bromo-α-ergocryptine (0.1 mg/day). Transplanted bovine mammary tissue was incubated for 4 hr in minimum essential medium containing 1 μCi/ml [³H]TdR. Two pieces were processed for autoradiography and the others were used for DNA assay and total [³H]TdR uptake. Bovine placental lactogen, prolactin, and growth hormone each increased [³H]TdR incorporation into DNA in a linear, dose-response manner. Addition of ovarian steroids to bPL resulted in a significant increase over protein hormones alone. Autoradiographic analysis indicated that the observed differences in DNA synthesis were due to hormonal effects on epithelial, rather than stromal, DNA synthesis. These results provide the first evidence of a mammogenic role of bovine placental lactogen.

The results of several recent studies have indicated that protein kinase C (PKC) may be involved in the prolactin (PRL) stimulation of mitogenesis in the Nb₂ node lymphoma cell line. The PKC activator 12-O-tetradeconylphorbol-13-acetate (TPA) at certain concentrations has been shown to potentiate the mitogenic effect of PRL, whereas at higher concentrations, TPA inhibits the PRL response. Several inhibitors of PKC have also been shown to impair the PRL stimulation of metabolic process in the Nb₂ cells. These studies provide further evidence for the likely involvement of PKC in the PRL stimulation of mitogenesis in the Nb₂ cells. A transient, time-dependent accumulation of PKC in the particulate fraction of the Nb₂ cells is observed in response to PRL. TPA is also shown to elicit a similar effect, albeit at a much earlier time and with a greater magnitude. On long-term exposure (3 days), high concentrations of TPA down-regulate the PKC enzyme; this down-regulation likely accounts for the inhibitory effect of high concentrations of TPA on the PRL stimulation of cell division. In further studies, the PKC inhibitors H-7 and gossypol were shown to inhibit the PRL stimulation of cell division in a concentration-dependent fashion.

In earlier studies, we demonstrated that continuous light (LL:LD, 24:0) stimulated tail regeneration wheras continuous darkness (DD:LD, 0:24) and pinealectomy depressed the same in the Gekkonid lizard, Hemidactylus flaviviridis, and, furthermore, exogenous prolactin significantly enhanced the regeneration process in lizards kept in 0:24 LD. However, the regeneration process in animals exposed to 24:0 LD was unaffected by the dopamine agonist, bromocriptine. This study with pimozide, an antipsychotic drug, and a potent dopamine receptor antagonist was conducted to ascertain whether the dopaminergic regulation of prolactin release is operative in lizards, as in mammals, and to provide further evidence for prolactin involvement in regenerative growth. Once daily intraperitoneal injection of 50 μg/kg pimozide to H. flaviviridis, 5 days prior to tail autotomy and 50 days thereafter, stimulated the regeneration process in lizards exposed to 0:24 LD. The initiation of regeneration, the total length of new growth (regenerate) produced by Day 50, and the total percentage replacement of the lost (autotomized) tails at the end of 50 days of experimentation were all significantly enhanced in pimozide-treated animals as compared with their counterparts injected with 0.6% sterile saline; in fact, better than saline-injected controls exposed to 24:0 LD of 638 lux intensity. The daily growth rate was also enhanced in pimozide-treated lizards. Interestingly, the pattern of regeneration as well as the final regenerate of pimozide-treated lizards were similar to those observed earlier in ovine prolactin-treated animals exposed to similar experimental photoperiodic schedules. The operation of the dopaminergic regulatory mechanism of prolactin release in lizards, as in mammals, is strongly indicated and the involvement of prolactin and its regulation by photoperiodism during lacertilian tail regeneration are discussed.

Logistic and biologic aspects of three separate means of administration of cyclopamine for experimental induction of terata or embryonic death in sheep were examined. Oral capsule administration of crystalline cyclopamine is logistically simple and biologically effective, but costly in terms of amount of compound required. Embryos were affected in five of seven ewes dosed cyclopamine orally at higher levels (four nonpregnant and one with cyclopia). Intramuscular administration of cyclopamine dissolved in ethanol was logistically simple but without biologic effect at levels tested. Three of three treated ewes had normal offspring. Osmotic minipump administration of powdered cyclopamine suspended in propylene glycol was logistically unsatisfactory with serious delivery complications. Osmotic minipump administration of cyclopamine dissolved in ethanol was logistically very satisfactory, and one ewe among three treated animals was nonpregnant at term. There were no nonpregnant ewes nor deformed offspring in 17 controls.

The relationship between β-endorphin(β-EP)/β-lipotropin(β-LP) and insulin secretion in the basal state and after glucose challenge was studied in obese male Zucker rats and their lean littermates. Baseline plasma β-EP/β-LP concentrations were similar in the two groups of animals. Baseline plasma insulin and serum glucose concentrations were significantly higher in the obese animals. Following glucose challenge, the increase in plasma β-EP/β-LP concentrations was significantly lower in the obese animals than in their lean littermates. Opioid blockade with naloxone failed to alter the baseline hyperinsulinemia and hyperglycemia seen in the obese animals. The data suggest that the hyperinsulinemia in the obese Zucker rat is not due to endogenous hyperendorp hinemia as shown in humans with polycystic ovary syndrome. The obese rats showed dissociation between glucose-stimulated plasma levels of β-EP/β-LP and insulin levels which may contribute to the hyperinsulinemia and insulin restance in these animals.

The purpose of this study was to determine whether a logistic regression model for the diagnosis of carpal tunnel syndrome (CTS) could be developed. Forty-eight variables were initially identified, for the 28 CTS and 34 non-CTS subjects, including 28 measures of nerve function, 6 anatomical measurements, 8 variables relating to disease symptoms, and 6 variables relating to physical attributes. An a priori clustering procedure was used to establish groups for the principal components analyses. The first principal component of each cluster was then used in a backward, stepwise logistic regression analysis. The best combination of candidate variables, as identified by the regression equation, was Raynaud's symptoms and median nerve motor function. The results of this study indicate that a model for CTS can be generated from a set of variables and that a linear combination of variables representing nerve function is closely associated with conduction decrements resulting from CTS.

The 3-hydroxypyridinium crosslinks of collagen were quantified in tissues of the skeleton and cardiovascular system of normal and copper-deficient rats. The copper-deficient rats used in this study displayed retarded growth, cardiac hypertrophy, anemia, and lowered liver copper concentrations. Quantification of the crosslinks by high performance liquid chromatography indicated that there were lower concentrations of collagen crosslinks in the hearts of copper-deficient animals, a finding that was manifest in both right and left ventricles. This was in contrast to the collagen of the aorta where no alteration in crosslink concentration was observed. The femoral diaphysis of copper-deficient rats also had lower amounts of collagen crosslinks than copper-supplemented animals, whereas crosslinking in the tibial diaphysis and articular cartilage was relatively unaffected by copper deficiency. These results are discussed with reference to the cardiac and skeletal abnormalities that occur in copper-deficient animals.

The effects of corn or menhaden oil and thyroxine treatment on hepatic mitochondrial respiration was studied. BHE rats were fed a 64% sucrose, 6% corn, or menhaden oil diet until they were 60–70 days of age. Succinate-supported mitochondrial respiration was studied at 3°C intervals from 4 to 40°C. Upper and lower activation energies and transition temperatures were determined through the calculation of Arrhenius plot. Menhaden oil plus daily thyroxine injection resulted in higher and lower activation energies than the other treatments. This combined treatment also resulted in lower state 3 and higher state 4 respiration rates and tighter coupling of respiration to ATP synthesis. These effects were thought to be due to the effect this treatment combination had on membrane fluidity.

Ethanol inhibits antibody-dependent cell-mediated cytotoxicity in a dose-dependent manner. The inhibitory effect of ethanol was reversed by the addition of excess calcium or calcium ionophore A23187. Excess calcium at 4–8 mM concentrations was required to reverse 50% of the inhibition caused by ethanol. In seven of nine experiments, 16 mM excess calcium completely reversed the inhibition and produced greater lysis than the control. Excess calcium in the absence of ethanol induced a dose-dependent increase in lytic activity by the spleen cells. However, the reversal of inhibition by ethanol could not be attributed to a simple additive effect resulting from the increased cytolytic capacity of the lymphocytes in the presence of excess Ca²⁺. Ionophore A23187 at 1 μm also partially reversed the inhibitory effect caused by ethanol. Ionophore alone did not potentiate lytic activity. When target cells were not sensitized with antibody, excess calcium had no effect on the lysis of target cells in the presence of ethanol-treated or untreated lymphocytes. These data suggest that ethanol inhibits antibody-dependent cell-mediated cytotoxicity at a calcium-dependent step.

Pancreatic exocrine secretion in the conscious rat is regulated by proteases secreted by the pancreas, and cholecystokinin (CCK) is known to be involved in its mechanism. It has also been reported that the absence of either pancreatic juice or bile in the duodenum could stimulate pancreatic secretion. Therefore, differences in CCK release responding to the exclusion of bile, pancreatic juice (PJ), or both bile and pancreatic juice (BPJ) from the intestine were examined by using a bioassay for cholecystokinin. Plasma CCK levels were increased by all three treatments compared with the basal value, the order of their effects being BPJ>PJ>bile diversion, and CCK concentrations produced by BPJ diversion were much greater than can be explained as simply summed effect of exclusions of bile and PJ. Pancreatic exocrine secretions were significantly increased by PJ and BPJ diversions, but the effect of bile diversion on the pancreas was not statistically significant. An additional infusion of CR-1409 (0.1 mg/rat), one of CCK receptor antagonists, inhibited exocrine function stimulated by BPJ diversion. We conclude (i) BPJ diversion is the strongest endogenous stimulant on CCK release; (ii) the potentiation between bile and PJ diversions is induced on CCK release; (iii) pancreatic protein secretion during BPJ diversion is mainly modulated by CCK.

We examined the responsiveness to prolactin and growth hormone of mammary epithelial cells from mice exposed neonatally to diethylstilbestrol (DES) and from control mice. The mammary epithelial cells were cultured inside collagen gels with serum-free medium containing insulin, epidermal growth factor, and linoleic acid. This produces prolactin-sensitive cells with low levels of casein production, as measured in cellular homogenates with a specific enzyme-linked immunosorbent assay for α-casein. The collagen gels containing these cells were then released and the medium supplements changed to insulin, linoleic acid, and prolactin at concentrations from 10 to 1000 ng/ml and growth hormone at 0, 10, or 100 ng/ml. This second phase of the culture, the differentiation phase, allows the cells to accumulate casein if they have this capacity. When cultured with prolactin only (no growth hormone), the cells from DES-exposed mice consistently accumulated 50–100% of the casein content of normal cells, but never more. Growth hormone, when added to prolactin-containing medium, increased casein accumulation above the levels seen with prolactin alone. Combinations of prolactin and growth hormone enhanced the difference between casein accumulation in DES-exposed and control cells, and DES-exposed cells were much less responsive to growth hormone. In our studies, the isolated mammary epithelial cells of estrogen-exposed mice are not more sensitive to prolactin than cells from normal animals as previous reports had suggested, but rather are generally less sensitive to hormonal stimulants.

The effects of ovariectomy and exogenous androgen administration on the indole and porphyrin metabolism of Syrian hamster Harderian glands were studied. Ovariectomy alone had no effect on any of the parameters analyzed. The administration of either testosterone or 5α-dihydrotestosterone increased the activity of N-acetyltransferase in the Harderian glands. However, androgen treatment failed to change the activity of hydroxyindole-O-methyltransferase. Melatonin content of the glands dropped 20 days after treatment with testosterone and 10 days after the administration of 5α-dihydrotestosterone. The porphyrin content of the Harderian glands was dramatically depressed after the administration of either androgen. It is concluded that the Harderian glands of Syrian hamsters are under an androgenic control involving 5α-dihydrotestosterone.

The tight skin mouse (tsk/+) is an autosomal dominant example of inherited fibrosis whose pathogenesis is unclear. Autoimmune phenomena have been described previously. This study demonstrates the adoptive transfer of skin fibrosis to lethally irradiated syngeneic +/+ recipients by the transplantation of both bone marrow and spleen cells. The recipient skin fibrosis was not associated with mast cell proliferation or degranulation as it is in the tsk/+ mouse, representing for the first time a dissociation between mast cells and fibrosis and introducing the potential for direct T cell-fibroblast interactions.

3'-Azido-3'-deoxythymidine (AZT) has attained wide clinical utility in the treatment of acquired immunodeficiency syndrome (AIDS). Unfortunately, associated with AZT use, is the development pf severe hematopoietic toxicity as manifested by anemia, neutropenia and overall bone marrow suppression. Interleukin-1 (IL-1), a cytokine, primarily produced by activated macrophages, has been involved in the control of hematopoiesis by acting synergistically with other hematopoietic growth factors, and has been demonstrated to be an effective agent in reducing the myelosuppression associated with the therapy for malignant disease. We report here the ability of recombinant human IL-1 alpha to protect normal murine hematopoietic progenitors (CFU-GM, BFU-E, and CFU-Meg) from the toxic effects of AZT. Following the determination of the LD5Q dose for each progenitor, IL-1 was added in co-culture studies (10 - 1000 units; 0.001 - 1.0 yg/ml protein) with adherent cell depleted marrow. Marrow progenitors expressed differences in AZT sensitivity, e.g., BFU-E, LD₅₀ 5 x 10′⁹M; CFU-Meg, LD₅₀ 10′⁷M; CFU-GM, 5 x 10′⁵M respectively. IL-1 inhibited AZT induced toxicity. The maximum IL-1 dose effect was observed for CFU-GM and CFU-Meg at 300 units, 0.3 yg protein; however BFU-E required a dose of 600 units, 0.6 yg/ml protein to reverse the effects of AZT. These results demonstrate marrow progenitors respond differently to AZT and identifies the potential efficacy of IL-I to minimize the hematopoietic toxicity associated with AZT treatment.

Xenopus oocytes were injected with size-fractionated mRNA isolated from the renal cortex of rabbit kidney and after 4 days incubation, PAH uptake in oocytes injected with mRNA (0.7 - 1.3 kb) was 8 to 45 fold that of the water injected controls. The oocyte to medium ratio of accumulated PAH was 1.95. The Km and Vmax for transport were 333 μM and 66.6 nmoles·oocyte⁻¹· min^−l, respectively. This Km is similar to that reported for PAH transport in intact kidneys and slices. The uptake of PAH was unaffected by the absence of Na⁺ or the presence of probenecid. Expression of the transport represents the first step in an effort to clone and identify the gene for PAH transport.