Potassium evoked catecholamine release from the nucleus tractus solitarius invitro

The release of endogenous catecholamines (CA) from rat brain slices containing the nucleus tractus solitarius (NTS) was measured using a sensitive radioenzymatic assay. KCl (35 to 75 mM) induced a dose-related increase in norepinephrine (NE) release. Dopamine (DA) release was maximal with 50 mM KCl. An increase in epinephrine (E) release was only observed with 75 mM KCl. NE and E release was totally calcium-dependent whereas DA release was only partially calcium-dependent. Subsequent administrations of KCl released less CA. The calcium dependency of the KCl induced release of E, NE, and DA suggests a neurotransmitter function in the NTS for these CA. A difference in storage sites and/or mechanisms may be responsible for the observed differences in sensitivity to KCl and to extracellular calcium.     

OKY-1581: A selective inhibitor of thromboxane synthesis invivo and invitro

OKY-1581 is an effective inhibitor of thromboxane synthesis $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. The generation of thromboxane B₂ (TxB₂), prostaglandin E (PGE) and prostaglandin F (PGF) was measured following clotting and during platelet aggregation induced by collagen. The presence of OKY 1581 either $\text{in}$$\text{vivo}$ or $\text{in}$$\text{vitro}$ caused a reduction in TxB₂ generation during clotting and platelet aggregation with a concomitant increase in PGE and PGF. The effect could be observed two hours after oral or subcutaneous administration of 5 to 100 mg per rabbit and lasted for 24 to 48 hours. The reduction in TxB₂ was not accompanied by an inhibition of clotting or platelet aggregation. OKY-1581 appears to be a suitable agent for studying the role of TxB₂ in atherosclerosis.     

Early steroid metabolism by chick blastoderm invitro

Yolk free blastoderms of chick embryo were incubated 3 or 22 hours with labeled pregnenolone, progesterone, 17-hydroxyprogesterone, dehydro-epiandrosterone, androstenedione, testosterone and estradiol-17β. Metabolites and unconverted substrates were found both in the incubation medium and in the cells. Enzymes responsible for identified conversions were: 17α-hydroxylase, 17-20-desmolase, Δ⁵3β- and 3α-hydroxysteroid dehydrogenase, 17β-hydroxysteroid dehydrogenase and 5α- and 5β-reductase. The results suggest that the steroid metabolizing enzyme activities found may reflect a more general ability of early embryonic cells.     

Effect of removing the zona pellucida on development of hamster and bovine embryos invitro and invivo

Uterine stage embryos collected from the hamster (8-cell) and cow (morula, early blastocyst) were monitored for development $\text{in}$$\text{vitro}$ (embryo culture) and $\text{in}$$\text{vivo}$ (embryo transfer) following premature removal of the zona pellucida.Removal of the zona pellucida did not significantly affect $\text{in}$$\text{vitro}$ development to the blastocyst stage of (1) 8-cell hamster embryos (zonae removed by a combined enzymic-mechanical procedure), (2) bovine morulae (zonae removed by mechanical means only) (3) early bovine blastocysts (zonae removed by the enzymic-mechanical technique).Zona-free hamster embryos formed significantly fewer viable fetuses than did zona-intact embryos. The lower incidence of fetal development observed following transfer of zona-free 8-cell hamster embryos may have resulted in part from the formation of chimeras by fusion of these embryos $\text{in}$$\text{utero}$. Such fusion was observed to occur $\text{in}$$\text{vitro}$ between zona-free embryos placed in close proximity. The proportion of pregnancies resulting from transfer of bovine blastocysts cultured from zona-free morulae was similar to that of zona-intact embryos.In this study we have demonstrated that (1) enzymic and mechanical procedures used to remove zonae pellucidae from uterine-stage hamster and bovine embryos do not adversely affect subsequent development of these embryos $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ and (2) zonae pellucidae are not required for normal development of these embryos. These findings have implications for microsurgery of mammalian embryos and for embryo transfer.     

Prostaglandin levels in preovulatory follicles from rabbit ovaries perfused invitro

Prostaglandin (PG) levels in follicular fluid from preovulatory follicles of rabbit ovaries perfused $\text{in}$$\text{vitro}$ were measured in order to compare PG changes in this model system with those that occur $\text{in}$$\text{vivo}$ and in isolated, LH-treated follicles $\text{in}$$\text{barvitro}$. One ovary from each rabbit was perfused without further treatment (control). The other ovary was exposed to LH (0.1 or 1 ug/ml) beginning 1 hour (h) after initiation of perfusion. Samples of perfusion medium were taken at frequent intervals for measurement of PGE, PGF, progesterone and estradiol 17β. The perfusions were terminated when the first ovulation occurred or appeared imminent as judged by changes in the size and shape of the follicles. Follicular fluid was then rapidly aspirated from all large follicles on both ovaries for PGE and PGF measurement.Ovulations occurred only in the LH-treated ovaries. Progesterone and estradiol levels were significantly elevated in the perfusion medium within 1 h of LH treatment in comparison to controls. PG levels in perfusion medium from the control and LH-treated ovaries were not different throughout perfusion and increased in both groups. In contrast, PG levels measured in follicular fluid from LH-treated ovaries were 4- to 5-fold greater than in fluid from control ovaries. It is concluded that ovulation induced by LH in this experimental model is accompanied by an increase in follicular PG levels similar to that seen in other $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$ models. This difference in follicular PG levels between the LH-treated and control ovaries is, however, not reflected in the perfusion medium.     

Glucosiduronidation and esterification of androsterone by human breast tumors invitro

The metabolism of ³H-androsterone was studied in homogenates (fortified with uridine 5'-diphosphoglucuronic acid and andenosine 3'-phosphate 5'-phosphosulfate) of eighteen breast tumors, one muscle underlying the primary breast carcinoma and metastatic axillary lymph nodes from a patient with suspected primary breast cancer. The major metabolites identified were less polar than androsterone. On saponification these lipoidal derivatives afforded androsterone as the only product (3 to 48%). Unmetabolized androsterone and lesser quantities of epiandrosterone, 5α-androstane-3α,17β-diol and 5α-androstane-3,17-dione comprised the free steroid fraction. Androsterone glucosiduronate was isolated (0.17–4.1%) from eight breast tumor homogenates and from the node tissue incubation (17%). There was no apparent correlation between glucuronyltransferase activity and histopathology or estrogen receptor content.     

An invitro assay system for chitin synthesis in insect tissue

The system described utilizes the abdominal integument of the grasshopper $\text{Melanoplus}$$\text{sanguinipes}$ as the tissue and radioactive glucose, glucosamine or UDP-N-acetyl glucosamine as substrate. A micromethod is described that makes it possible to use as little as 2 μl total volume of incubation. After an initial lag period, incorporation rates become linear for at least 2 hrs. Cell disruption leads to immediate and complete loss of activity.     

Effect of organic acid on gastrointertinal motility of rat invitro

The quantity of organic acids ( lactic acid, acetic acid, propionic acid and butyric acid ) in the content of the gastrointestinal tract of germ-free and conventional rats and the $\text{in}$$\text{vitro}$ effects of the organic acid on the motility of the gastrointestinal tract of rats were investigated.Organic acids were detected only in the gastrointestinal contents of conventional rats but not in those of germ-free rats.Lactic acid detected in the stomach of rats stimulated the motility of both small and large bowel while acetic acid, propionic acid and butyric acid found in the cecum stimulated the motility of the large bowel but not of small bowel.     

The ionophore X537A (Lasolocid) transiently increases acetylcholine release from rat brain invitro

The effect of X537A on acetylcholine (ACh) release was examined $\text{in vitro}$ in superfused slices of rat cerebrum and striatum. The ionophore (30 μM) induced a transient release of ACh which was not dependent on calcium in the medium. Also in contrast to K⁺-stimulated release, X537A-induced release was not sustained by 10^?5M choline in the superfusion medium and not inhibited by 5 × 10^?4M pentobarbital. The ionophore did not transport ACh or choline from an aqueous to an organic phase. Both K⁺ and X537A inhibited 1 μM (³H) choline uptake into striatal synaptosomes but this effect of X537A was more extensive and less reversible than that caused by K⁺. X537A did not inhibit choline acetyltransferase activity.     

Luteinizing hormone stimulates the production of prostacyclin by isolated ovarian cell invitro

Granulosa cells isolated from mature Graafian follicles of swine produced significant quantities of immunoreactive 6-keto-PGF₁α under chemically defined conditions in vitro. Luteinizing hormone elicited a dose-dependent stimulation of 6-keto-PGF₁α accumulation, but follicle stimulating hormone, prolactin, L-epinephrine, estradiol-17B, or PGE₂ were devoid of effect. The time-dependent in vitro production of 6-keto-PGF₁α by ovarian cells was susceptible to inhibition by indomethacin, U-51506, cycloheximide, and actinomycin D. These observations implicate granulosa cells in the specific and hormonally regulated production of prostacyclin.     

Dopamine release induced by electrical field stimulation of rat hypothalamus invitro: Inhibition by melatonin

Microdissected slices of rat hypothalamus were incubated with ³H -dopamine and then subjected to two successive sets of electrical field stimulation in a superfusion chamber. Neurotransmitter release was found to be calcium dependent and the amount of release was determined by scintillation counting of the effluent buffer. The release obtained following the first train of stimuli served as an internal reference. The samples were exposed to drugs during the interval between the two sets of stimuli. Using this technique, as well as K⁺-evoked depolarization, we were able to show that subnanomolar concentrations of melatonin, the hormone secreted from the pineal gland, inhibits dopamine release from hypothalamic slices. The possibility that melatonin modulates neurotransmission in the brain is therefore indicated.     

Actions of a nicotinic agonist,DMPP, on intestinal ion transport invitro

High-dose carbachol (10^?3 M) has previously been shown to cause NaCl absorption in short-circuited rabbit ileum. The mechanism of this effect may be norepinephrine release induced by carbachol activation of presynaptic nicotinic receptors on adrenergic neurons. Norepinephrine then interacts with postsynaptic α-adrenergic receptors on intestinal mucosal cells to stimulate neutral NaCl absorption and inhibit electrogenic bicarbonate secretion. The present paper examines the $\text{in vitro}$ intestinal ion transport effects of DMPP an agent which is more specific than carbachol on nicotinic cholinergic receptors. DMPP (10^?5 M) caused a transient increase followed by prolonged depression of the short-circuit current, increased NaCl absorption and increased tissue conductance. This effect was antagonized by hexamethonium and phentolamine. It is concluded that nicotinic cholinergic agents stimulate norepinephrine release from adrenergic nerves and effect intestinal ion transport just as norepinephrine does.     

Bromobenzene and p-bromophenol toxicity and covalent binding invivo

A hepatotoxic dose of bromobenzene (3 mmoles/kg) decreases hepatic glutathione concentration in rats by approximately 80% within 5 hr following ip injection. A major bromobenzene metabolite, p-bromophenol at a similar dose did not significantly alter hepatic glutathione levels compared to controls. Twenty four hr after administration, serum glutamate pyruvate transaminase (SGPT) levels were significantly increased by bromobenzene but not by p-bromophenol. After ¹⁴C-bromobenzene administration, a significant amount of covalently bound radiolabel was detected in liver, kidney and small intestine. A small amount of covalently bound radiolabel was also detected in the lung. After a similar dose of ¹⁴C-bromophenol, covalently bound radiolabel was found in liver (62% of the amount detected with ¹⁴C-bromobenzene) and smaller amounts were detected in kidney, small intestine and lung. These data are consistent with the view that the hepatotoxity and glutathione depleting ability of bromobenzene are mediated mainly by bromobenzene-3, 4-oxide rather than by chemically reactive metabolites of p-bromophenol derived from bromobenzene. Covalently bound radiolabel from ¹⁴C-bromobenzene, however, may be derived from both bromobenzene-3, 4-oxide and the nontoxic reactive metabolites of p-bromophenol.     

The estrogenic activity of DDT: Invivo and invitro induction of a specific estrogen inducible uterine protein by o,p'-DDT

The pesticide o,p'-DDT stimulates the production of a specific uterine protein, the so-called induced protein or IP, normally associated with an estrogenic response of the uterus. $\text{In}$$\text{vivo}$ stimulation of IP production is observed 1 hour after the administration of 250 mg/kg of o,p'-DDT to immature rats. $\text{In}$$\text{vitro}$ stimulation of IP production is observed after a 1 hour incubation of uteri with 100 μM o,p'-DDT. This $\text{in}$$\text{vitro}$ response is blocked by Actinomycin D. In contrast to o,p'-DDT, which binds to the cytoplasmic estrogen receptor and stimulates IP production, p,p'-DDT which does not bind well to the estrogen receptor does not stimulate IP production $\text{in}$$\text{vitro}$. These findings represent the first report of an estrogenic effect of o,p'-DDT in a completely $\text{in}$$\text{vitro}$ system.     

Invitro effect of d-lysergic acid diethylamide on immunoglobulin synthesis

Rabbit anti-fluorescyl antibody producing lymphoid cells incubated $\text{in}$$\text{vitro}$ with LSD do not secrete the 7S form of immunoglobulin. The low molecular weight extracellular labeled material shows no measurable anti-fluorescyl antibody activity. Results indicate that during a short incubation period LSD interferes with tryptophan incorporation into antibody protein.     

Invitro evaluation of granulocyte differentiation of diffusion chamber inocula

The semi-soft agar colony assay permits an $\text{in}$$\text{vitro}$ analysis of committed myeloid stem cell (CFU-c) proliferation capacities. In this paper this procedure has been used in combination with prior diffusion chamber culturing to determine the effect of host influences upon this committed stem cell population. This “double-seeding” procedure of first culturing bone marrow cells in diffusion chambers and then re-seeding them in agar furnishes data suggesting a relationship between $\text{in}$$\text{vivo}$ diffusion chamber transitional lymphocytes and $\text{in}$$\text{vitro}$ CFU-c seeding capacities. Diffusion chamber culturing offers a means of monitoring granulopoiesis and selects for enrichment of stem cell numbers. Detection and quantification of diffusion chamber stem cell enrichment is easily assessed by seeding chamber contents into the agar colony assay.     

Pharmacology of excitatory amino acid receptors mediating the stimulation of rat cerebellar cyclic GMP levele invitro

L-glutamate and related amino acids produced dose-related increases in cyclic GMP concentrations in cerebellar slices from 8-day old rats. The effects of L-glutamate, L-aspartate, and N-methyl-D-aspartate were antagonised in part by glutamate diethylester, while D-α-aminoadipate and DL-α-aminosuberate were ineffective. Kainic acid, although producing an effective stimulation of cyclic GMP, failed to achieve the same maximal response as glutamate, suggesting different sites of action. Cyclic GMP levels were also increased, although to a lesser extent, by L-glutamate in slices from hippocampus, medulla, hypothalamus, cortex, striatum and spinal cord of young animals. In the adult, however, this response was no longer observed.     

Guanylate cyclase from Dictyosteliumdiscoideum

Guanylate cyclase from crude homogenates of vegetative $\text{Dictyostelium}$$\text{discoideum}$ has been characterized. It has a pH optimum of 8.0, temperature optimum of 25°C and requires 1 mM dithiothreitol for optimal activity. It strongly prefers Mn⁺⁺ to Mg⁺⁺ as divalent cation, requires Mn⁺⁺ in excess of GTP for detectable activity, and is inhibited by high Mn⁺⁺ concentrations. It has an apparent Km for GTP of approximately 517 μM at 1 mM excess Mn⁺⁺.The specific activity of guanylate cyclase in vegetative homogenates is 50–80 pmoles cGMP formed/min/mg protein. Most of the vegetative activity is found in the supernatant of a 100,000 x g spin (S100). The enzyme is relatively unstable. It loses 40% of its activity after 3 hours storage on ice. Enzyme activity was measured from cells that had been shaken in phosphate buffer for various times. It was found that the specific activity changed little for at least 8 hours. Cyclic AMP at 10^?4 M did not affect the guanylate cyclase activity from crude homogenates of vegetative or 6 hour phosphate-shaken cells.