The inotropic action of adrenergic agonists on the heart ventricles of the chick embryo]

The effects of adrenergic drugs on the twitch tension of the electrically driven (1.2-1.5 Hz) ventricular preparations from 2-20-day old chick embryos and hatched chicks were studied. Agonists evoked positive inotropic responses of 3-day embryonic ventricles and of ventricles from older animals. 2-day embryonic ventricles were unresponsive. 5-day embryonic ventricles were most sensitive to agonists (EC50 value of adrenaline = 4.5 x 10(-9) M), while ventricles from 14-20-day old embryos had a minimal sensitivity (1-2 x 10(-9) M), while ventricles from 14-20-day old embryos had a minimal sensitivity (1-2 x 10(-7) M). The order of agonists activity (isoproterenol greater than noradrenaline greater than adrenaline much greater than phenylephrine) and the high potency of propranolol as antagonist of adrenaline indicate that responses are mediated with beta-adrenoceptors. The role of GTP-binding protein for the regulation of adrenoreactivity in embryonic chick heart during ontogenesis is discussed.     

Differential responsiveness to replacement therapy of pre- and postpubertally castrated mice with respect to intermale aggression

Male mice castrated before Day 6 of postnatal life differ from adult castrates in that they do not exhibit normal patterns of intermale aggression following replacement therapy as adults. Two experiments sought to determine the effect castration after Day 6, but before puberty, would have on this response to adult replacement therapy. It was found that adult castrates showed some increases in fighting as early as six hr after a subcutaneous (s.c.) injection of 2 mg testosterone propionate (TP). At 30 hr after treatment 14/15 mice fought, while at 72 hr 15/15 fought and the number of fights in 10 min was significantly higher than at 30 hr. In the second experiment, the response to adult treatment with 2 mg TP (s.c.) in mice castrated in Day 10 or Day 50 was compared. Different groups were tested at 16, 40, 64, and 88 hr after TP treatment. The latency to respond to TP was significantly less and the level of fighting obtained was significantly greater in Day 50 castrates than in Day 10 castrates at each time of testing. The proportion of Day 10 (14/15) and Day 50 (15/15) castrates fighting was equal by 88 hr, while the mean fighting frequency was significantly lower in Day 10 castrates (M = 4.93 ± 4.78) than in Day 50 castrates (M = 8.06 ± 1.63). Day 50 castrates fought significantly more often than controls at 40 hr, whereas the level of fighting attained by Day 10 castrates was not significant until 64 hr. These results suggest that even after the organizational period for aggression, testosterone is necessary for maintenance and/or preparation of the substrate essential to the elicitation of aggression.     

Role of divalent cations in the genesis of the frog electro-olfactogram

In frogs, underwater electro-olfactograms (EOGs) in response to 10(-5) M isoamyl acetate were recorded under restricted cationic environments. The olfactory mucosa was superfused by sodium-free sucrose solutions, and the effect of the addition of millimolar concentrations of calcium, cobalt, barium, or cadmium was studied. Only calcium and barium restored the response; sodium and cobalt were less efficient. Cobalt did not alter the restoring effect of calcium. Cadmium resulted in irreversible blocking irrespective of the presence of calcium. These results are discussed in terms of possible ionic mechanisms mediating olfactory responses.     

Endotrophic calcium, strontium, and barium spores of Bacillus megaterium and Bacillus cereus

Foerster, Harold F. (The University of Texas, Austin), and J. W. Foster. Endotrophic calcium, strontium, and barium spores of Bacillus megaterium and Bacillus cereus. J. Bacteriol. 91:1333-1345. 1966.-Spores were produced by washed vegetative cells suspended in deionized water supplemented with CaCl(2), SrCl(2), or BaCl(2). Normal, refractile spores were produced in each case; a portion of the barium spores lost refractility and darkened. Thin-section electron micrographs revealed no apparent anatomical differences among the three types of spores. Analyses revealed that the different spore types were enriched specifically in the metal to which they were exposed during sporogenesis. The calcium content of the strontium and the barium spores was very small. From binary equimolar mixtures of the metal salts, endotrophic spores accumulated both metals to nearly the same extent. Viability of the barium spores was considerably less than that of the other two types. Strontium and barium spores were heat-resistant; however, calcium was essential for maximal heat resistance. Significant differences existed in the rates of germination; calcium spores germinated fastest, strontium spores were slower, and barium spores were slowest. Calcium-barium and calcium-strontium spores germinated readily. Endotrophic calcium and strontium spores germinated without the prior heat activation essential for growth spores. Chemical germination of the different metal-type spores with n-dodecylamine took place at the same relative rates as physiological germination. Heat-induced release of dipicolinic acid occurred much faster with barium and strontium spores than with calcium spores. The washed "coat fraction" from disrupted spores contained little of the spore calcium but most of the spore barium. The metal in this fraction was released by dilute acid. The demineralized coats reabsorbed calcium and barium at neutral pH.     

The binding of cations to the surfaces of cells from early chick blastoderms

Cells of the early chick blastoderms are either preparing for or undergoing regulated morphogenetic movements which culminate in the formation of a three-layered embryo. Information on the changes in the physical-chemical properties of cell surfaces may help in the understanding of this process. The binding of magnesium, manganese, strontium, barium and lanthanum to surfaces of early embryonic cells was estimated by the changes induced by these cations in the cells' electrophoretic mobilities (EPM). Cells show a positive EPM at concentrations of MgCl2 and MnCl2 at 3 X 10(-2) M while SrCl2 and BaCl2 were not able to reverse the cells' charge at concentrations up to 6 X 10(-2) M. CaCl3 reversed the cells' EPM at concentrations as low as 5 X 10(-3) M. Our results suggest that the surfaces of early embryonic cells have a high affinity for Mg and Mn. This is indicated by a reversal of polarity which cannot be detected in cells of differentiating or adult tissues at the cation concentrations used in these experiments.     

Stimulation with LH of progesterone production by rabbit corpora lutea in vitro

Quartered CL from 7-day pseudopregnant rabbits were incubated at 37 degrees C for 0-180 min in the presence of BSA, LH or adrenaline in Krebs-Ringer-bicarbonate buffer. Total progesterone at each time point was quantified in homogenates of tissue plus incubation media and expressed relative to CL protein. Progesterone increased linearly with time during the first 30 min of incubation in the presence of BSA. LH and adrenaline markedly accelerated progesterone accumulations relative to the BSA control. At 10 min, progesterone accumulation in the presence of LH and adrenaline were 2.4 and 5.9 times that in the absence of stimulators, respectively. Both hormones caused concentration-dependent increases in progesterone and the apparent ED50 was 0.75 microgram/ml for LH and adrenaline. The CL obtained from ovaries of 7-day pseudopregnant rabbits are therefore capable of an acute steroidogenic synthetic response to LH as well as adrenaline.     

Hormonal control of serving capacity in bulls

The hormonal control of serving capacity in bulls was investigated in four sets of identical twin bulls averaging 9.9 ± 1.8 (SD) (H), 4.1 ± 1.0 (M1), 4.3 ± 1.0 (M2) and 1.9 ± 1.0 (L) services in four standardised tests. Within each pair, twin brothers had similar serving capacity. One bull from each twin set was castrated while its brother remained entire. When the castrates were maintaining their nadir in serving capacity (4, 0, 1 and 0) services for H, M1, M2 and L respectively) each was given testosterone propionate (TP) (1 mg/kg body weight) at 4 to 5 day intervals for 3 weeks, and along with its entire twin brother, tested for serving capacity 1 day after TP administration. Serving capacity of each castrate returned to its high, medium or low precastrational level and to the contemporary level of its entire twin brother. The plasma testosterone levels of the H, M1 + M2 and L entire bulls were similar (16.0 ± 3.0, 16.0 ± 3.0 and 22.4 ± 3.0 ng/ml respectively) despite large differences in serving capacity. It was concluded that (i) the presence of testosterone above a threshold level (<7 ng/ml in the assay used) is necessary for the maintenance of serving capacity and (ii) differences in serving capacity between bulls are not due to differences in plasma testosterone levels but to differences in their somatic responsiveness to threshold levels of testosterone.     

Effect of neonatal castration on sex steroid receptor levels in the hypophysis of male rats

The content of estradiol and testosterone cytosolic and nuclear receptors has been studied in the pituitary body of adult male rats gonadectomized on day 1-3 after birth (long-term castrates) or in adulthood (short-term castrates). Intact male rats and long- and short-term castrates had the same level of cytosolic and nuclear estrogen receptors. The number of cytoplasmic and nuclear testosterone-binding sites was identical in the pituitary body of adult intact mice and long-term castrates. Contrastingly, the concentrations of androgen cytosolic and nuclear receptors were significantly lower in neonatally castrated males compared to intact adult animals. The results obtained indicate that nuclear testosterone receptors in the pituitary body mediate negative feedback effect of androgen on the release of luteinizing hormone and that the formation of thin mechanism occurs within the first days of life.     

Effects of androgens on body weight, feeding, and courtship behavior in the pigeon

Adult male pigeons, some intact and some castrated in adulthood, were housed in individual cages kept in an isolated room with temperature and lighting controlled. Weekly measurements were made of ad lib. food intake and body weight for 4 mo after surgery. Castration was followed by a significant depression in body weight and by initially depressed but then progressively enhanced feeding. Food deprivation elicited an increase in food intake proportional to body weight loss, but castrates consumed less food at 100%, 90%, and 80% of ad lib. feeding weight than either intact birds or castrates treated daily with testosterone propionate (TP). Castrates gained weight and ate more than controls in response to daily treatments (im) with TP (6 mg/400 g) or 5a-dihydrotestosterone (DHT, 6 mg/400 g), while androstenedione (15 mg/400 g) and androsterone (15 mg/400 g) were ineffective. Administration of 100 mg DHT (sc) to castrates produced a significant enhancement of body weight without elevating the level of food intake. The biological potency of these diverse androgens on male courtship behavior was reciprocal to that for weight-promoting potency. The results suggest that the structural requirements of the androgen molecule for promoting body weight differ from those for stimulating sexual behavior.     

Pharmacological actions of the calcium antagonist propyl-methylenedioxyindene in skinned vascular smooth muscle

Previous studies provided strong evidence that propyl-methylenedioxyindene (pr-MDI) interfered with calcium at an intracellular site. To further characterize the mechanism of action of pr-MDI, its pharmacological actions on chemically skinned vascular smooth muscle were examined. Rat caudal artery strips were chemically skinned with saponin (0.15 mg/mL for 1 h). The efficiency of the skinning was evidenced by a loss of contractile response to 74 mM K+. The intactness of the regulatory and contractile proteins was ascertained by the ability of the skinned tissue to contract in response to Ca2+ (free Ca2+ concentration of 10(-4) or 10(-6)M). Caffeine (25 mM) induced contraction was used as an index of the functional integrity of the sarcoplasmic reticulum in the skinned preparations. Contraction of the skinned artery with a free Ca2+ concentration of 10(-6)M was significantly obtunded by 1 X 10(-4)M trifluoperazine (a calmodulin antagonist) but not by 1 X 10(-4)M pr-MDI. Contraction of the skinned artery evoked by 25 mM caffeine in the absence of extracellular calcium was significantly obtunded by 1 X 10(-4)M pr-MDI but not by 1 X 10(-6)M nifedipine (a calcium channel blocker). The results indicate that pr-MDI acts intracellular to block calcium mobilization from the sarcoplasmic reticulum without directly interfering with the regulatory and contractile proteins.     

Hormonal regulation of preputial gland function in male Microtus montanus, the montane vole

1. Preputial gland function in male Microtus montanus is androgen-dependent, both in terms of preputial weights and in the production of a series of lipids which are present in M. montanus and absent from Microtus pennsylvanicus. 2. Production of these species-typical lipids is decreased but not eliminated in castrates, as well as in adrenalectomized castrates treated with corticosterone. Therefore, in the total absence of androgens, a low level of these lipids is still produced. 3. 5 alpha-dihydrotestosterone and 17 beta-estradiol enanthate have limited effects on maintenance of preputial weight, suggesting that testosterone itself is the active steroid in the preputial gland of this species.     

Barium promotes anchorage-independent growth and invasion of human HaCaT keratinocytes via activation of c-SRC kinase

Explosive increases in skin cancers have been reported in more than 36 million patients with arsenicosis caused by drinking arsenic-polluted well water. This study and previous studies showed high levels of barium as well as arsenic in the well water. However, there have been no reports showing a correlation between barium and cancer. In this study, we examined whether barium (BaCl(2)) may independently have cancer-related effects on human precancerous keratinocytes (HaCaT). Barium (5-50 μM) biologically promoted anchorage-independent growth and invasion of HaCaT cells in vitro. Barium (5 μM) biochemically enhanced activities of c-SRC, FAK, ERK and MT1-MMP molecules, which regulate anchorage-independent growth and/or invasion. A SRC kinase specific inhibitor, protein phosphatase 2 (PP2), blocked barium-mediated promotion of anchorage-independent growth and invasion with decreased c-SRC kinase activity. Barium (2.5-5 μM) also promoted anchorage-independent growth and invasion of fibroblasts (NIH3T3) and immortalized nontumorigenic melanocytes (melan-a), but not transformed cutaneous squamous cell carcinoma (HSC5 and A431) and malignant melanoma (Mel-ret) cells, with activation of c-SRC kinase. Taken together, our biological and biochemical findings newly suggest that the levels of barium shown in drinking well water independently has the cancer-promoting effects on precancerous keratinocytes, fibroblast and melanocytes in vitro.     

The role of calcium channels in substance P-induced contractile response in the rat iris

This study was undertaken to assess the role of calcium channels in the contractile response induced by substance P in the isolated rat iris. Substance P produced graded and sustained contraction in the rat iris. Pre-incubation of preparations with thapsigargin (1 μM), verapamil (1 μM), isradipine (1 μM) or with ω-conotoxin MCIIA (0.1 μM) did not significantly inhibit substance P-mediated contraction in the isolated rat iris. However, pre-incubation of the preparations with nicardipine (1 μM) or ruthenium red (1 mM) caused parallel displacement to the right of the substance P concentration–response curve without affecting its maximal response. In contrast, amiloride (1 μM), markedly inhibited substance P-mediated contraction (73±5%), while econazole (1 mM) also significantly inhibited (44±11%) substance P-mediated contraction in the isolated rat iris. Collectively, these results suggest that substance P-mediated contractile response in the isolated rat iris depends largely on the influx of external Ca²⁺, by a mechanism which might involve the T-type calcium channels.     

Pharmacological actions of a new class of neuropeptides, the leucokinins I-IV, on the visceral muscles of Leucophaea maderae

1. Leucokinins I-IV did not activate visceral muscles uniformly as a class but rather showed a selective action on the muscles of the hindgut. This organ showed a contractile response to all of the leucokinins at 3 x 10(-10) M that was 5-10% above the mean level of spontaneous activity. The maximum response for each peptide was recorded at 2.1 x 10(-7) M. 2. Both the foregut and the oviduct were 100-1000 fold less sensitive than the hindgut, and each of the former organs required more than 10(-8) M to elicit a detectable excitation. The heart, by comparison, failed to give consistent responses with any of the peptides. 3. The leucokinins caused a protracted excitation of contractile events in the hindgut that lasted for more than 60 min. Moreover, all four peptides evoked contractions from hindguts after membrane depolarization with 158 mM potassium. 4. This result shows that nonsynaptic receptors for the peptides exist in visceral muscle. The leucokinins showed no evidence of facilitating the reentry of calcium into calcium depleted hindgut preparations.     

Nicotine and cotinine inhibit steroidogenesis in mouse Leydig cells

Cigarette smoking alters plasma testosterone concentrations in men. The objectives of this study were to determine if nicotine and cotinine, two alkaloid products of cigarettes, affect luteinizing hormone(LH)-stimulated steroidogenesis in isolated adult mouse Leydig cells. Leydig cells from adult Swiss-Webster mice were isolated by linear density gradient and incubated (95% O2, 5% CO2) in minimum essential medium at 37 C for 3 hours with LH (10 ng) and with or without nicotine or cotinine (10(-5)-10(-7) M). Both nicotine and cotinine produced dose response inhibition (P less than 0.05) of LH-stimulated testosterone production (50-70%). The addition of 8-bromo-3',5'-cyclic monophosphate (cAMP, 500 uM) stimulated steroidogenesis comparable to LH in the absence of the alkaloids, but both nicotine and cotinine significantly (P less than 0.05) reduced testosterone production in response to cAMP, suggesting that the alkaloids inhibit testosterone production in response to LH distal to the formation of cAMP. In MEM without calcium, LH-stimulated testosterone synthesis was decreased, and neither nicotine nor cotinine significantly affected steroidogenesis. The addition of a calcium ionophore in MEM with normal calcium content enhanced (P less than 0.05) the inhibitory effects of nicotine and cotinine on LH-responsive steroidogenesis. A calcium channel blocking agent, verapamil, at 10uM significantly (P less than 0.05) reversed the inhibition of LH-stimulated testosterone production produced by both alkaloids when incubated in the medium with a normal calcium concentration. These results suggest that nicotine and cotinine either affect intracellular calcium content or block the effects of calcium on steroidogenesis in mouse Leydig cells.     

Ultrastructure of normals and castrates and the effects of testosterone and ultraviolet (UVL-B) irradiation on scrotal skin of rats.

The ultrastructure of the testosterone dependent epidermal melanocyte system of the scrotal skin of normals and castrates, with and without testosterone replacement therapy, and UVL-B (280-315 nm) radiation in black Long Evans rats is reported. UVL-B increases melanocyte activity, melanosome forming apparatus, (size of Golgi zone and RER, and quantity of cytoplasmic vesicles, dendrites, and stages of melanosomes) in normals and in castrates. Testosterone replacement therapy to castrates is not a prerequisite for stimulation by UVL-B, but it enhances the effects of UVL-B without restoring normalcy as melanosome packaging into complexes predominates. After UVL-B stimulation of normals or castrates, melanocyte dendrites are observed more often. Melanocyte dendrites of skin of castrated rats are observed less often than in normals, but with testosterone replacement therapy, the dendrites become more numerous. Melanosomes donated to keratinocytes are mostly located as singles in normals and as complexes in castrates. After UVL-B, castration, or testosterone replacement therapy, the melanosomes are packaged in keratinocytes in complexes larger than in normals. In the epidermis of long term castrates (9-109 days), non-specific clear cells are observed and Langerhans cells containing melanosomes; we did not observe them in normals. Melanocytes of castrates have a reduced melanosome forming apparatus. The dermis of castrates contains many dermal melanocytes in the superficial dermis with melanosomes in several stages of formation. These cells are not apparent in normals at this location in the dermis. Testosterone replacement therapy and/or UVL-B administered to castrates does not restore the epidermal melanocyte system nor the dermis to precastration ultrastructural appearance; castration has a permanent altering effect as melanosomes are packaged into complexes.     

Rapid androgen actions on calcium signaling in rat sertoli cells and two human prostatic cell lines: similar biphasic responses between 1 picomolar and 100 nanomolar concentrations

Androgen-induced calcium fluxes and gap junctional intercellular communication (GJIC) were studied in three different cell types. A transient (2-3 min duration) increase in intracellular calcium levels was observed within 20-30 sec of androgen addition, which was followed by a plateau phase with steroid concentrations higher than 1 nM. The kinetics of the calcium responses were similar in immature rat Sertoli cells, which contain normal nuclear receptors; the human prostatic tumor cell line, LNCaP, which contains a mutated nuclear receptor; and the human prostatic cell line, PC3, which does not contain a nuclear receptor. The human A431 tumor cell line did not respond to androgens. Concentrations of testosterone and the synthetic androgen, R1881, between 1-1000 pM induced transient calcium increases with ED(50) values near 1 pM and 1 nM, whereas dihydrotestosterone (DHT) was not active at these concentrations. At concentrations higher than 1 nM, testosterone, R1881, and DHT were equipotent in stimulating an increase in calcium that lasted for more than 10 min, with ED(50) values between 5 and 20 nM. Testosterone covalently bound to albumin was also active, whereas 11 related androstane compounds as well as progesterone and estradiol-17beta were inactive at 1000 nM. The calcium response induced by the three androgens (10 nM) was abolished in all cell types by hydroxyflutamide (1000 nM) and finasteride (1000 nM), but not by cyproterone acetate (1000 nM). The calcium response was also abolished in the absence of extracellular calcium and strongly inhibited by the presence of verapamil. Exposure of the responsive cells to brief (150-sec) pulses of androgens generated calcium responses that were similar to those after continuous exposure. After exposure of Sertoli cells for only 30 sec to 100 nM testosterone, the calcium response lasted for at least 50 min. Although nuclear binding of androgens could be demonstrated, there was no evidence for tight binding to the plasma membrane under similar conditions. When protein synthesis was inhibited, an enhancement of GJIC between rat Sertoli cells, but not between LNCaP cells or PC3 cells, was observed within 15 min of the addition of 10 nM testosterone. Because nuclear androgens are not present in PC3 cells and many functional properties of the responsive system are different from the nuclear receptor in all three cell types, we postulate the existence of an alternative cell surface receptor system with biphasic response characteristics (high and low affinity). The calcium signals are probably coupled to the regulation of gap junctional efficiency between Sertoli cells. The low-affinity receptors may convey complementary androgen signals at elevated local levels such as in the testis, when nuclear receptors are (over)saturated.     

Intracellular calcium homeostasis during hydrogen peroxide injury to cultured P388D1 cells

The effects of exposure of cultured P388D1 cells to H2O2 on intracellular free calcium ([Ca++]i) was investigated utilizing the intracellular fluorescent calcium chelator "Quin 2." [Ca++]i rose from approximately 150 nM to greater than 2 microM over a time course that was strongly dependent on the concentration of H2O2 used (5 X 10(-5) to 5 X 10(-3) M). After exposure of P388D1 cells to 5 X 10(-3) M H2O2, Quin 2 was fully saturated between 15 and 30 min exposure. During this time, no apparent change in the rate of equilibration of 45Ca++ from the extracellular medium could be detected, whereas in cells preloaded with 45Ca, net 45Ca was lost from the cells at a greater rate than controls. Measurements of total cellular calcium by atomic absorption spectroscopy confirmed that there was a net loss of calcium from the cells during the first 30 min. At time points greater than 45 min after exposure to H2O2 the influx of extracellular 45Ca and net intracellular Ca++, Na+ and K+ rapidly increased. Half times for H2O2 catabolism by the cells varied from about 8 min at 5.0 X 10(-4) M H2O2 to 14.0 min at 5.0 X 10(-3) M. When the total [Ca++]i-buffering capacity of the Quin 2 pool was varied by increasing the loading of intracellular Quin 2 by 68-fold (1.1 X 10(2) - 7.6 X 10(3) amol per cell), the rate of rise of [Ca++]i was depressed by only 1.6-fold following exposure to 5 mM H2O2. During the rise of intracellular [Ca++]i, cell morphology was observed by both light and scanning electron microscopy and revealed that "surface blebs" appeared during this phase of injury. Both the rise in [Ca++]i and "blebbing" were observable before any loss in cell viability was detected by either loss of Trypan blue exclusion or loss of preloaded 51Cr from the cells. From these results we conclude the following, H2O2 exposure induces a dose-dependent disturbance of intracellular calcium homeostatis; the rise in [Ca++]i is mediated by exposure to H2O2 in the early phase of the injury, and is not dependent on the continuing presence of the oxidant; the rate of rise of [Ca++]i is largely independent of the quantity of calcium mobilized to the Quin 2 pool; during the early phase (less than 30 min) of rise of [Ca++]i, only intracellular calcium is involved in the response; these events occur concomitantly with gross morphological changes to the plasma membrane.(ABSTRACT TRUNCATED AT 400 WORDS)