Effect on an antagonistic analog of gonadotropin releasing hormone upon ovarian granulosa cell function.

We have recently demonstrated that gonadotropin releasing hormone (GnRH) acts directly on ovarian granulosa cells to inhibit the follicle stimulating hormone (FSH)-induced increase in granulosa cell steroidogenesis $\text{in}$$\text{vitro}$. A GnRH antagonist, [D-pGlu¹, D-Phe², D-Trp^3,6] GnRH (A), which is known to antagonize GnRH-stimulated gonadotropin release by cultured pituitary cells, was tested in the granulosa cell system. GnRH (10^?8M) inhibited estrogen and progesterone production by FSH-treated granulosa cells $\text{in}$$\text{vitro}$, whereas the antagonist A (10^?6M) did not affect FSH stimulation of steroidogenesis. Antagonist A, when added together with GnRH and FSH, blocked the GnRH inhibition of FSH-induced steroidogenesis. Estrogen and progesterone production by granulosa cells was increased by 50% at a molar ratio (IDR₅₀) of $\text{20}\text{1}\text{and}\text{12}\text{1}$ ([antagonist]/[GnRH]), respectively. At 10^?6M, antagonist A completely prevented the GnRH (10^?8M) inhibition. A similar effect of antagonist A was seen in FSH-induced increase of luteinizing hormone (LH) receptor content. FSH treatment for 2 days $\text{in}$$\text{vitro}$ induced an 8-fold increase in LH receptor content in cultured granulosa cells; concomitant treatment with 10^?8M GnRH completely inhibited the FSH effect. Antagonist A (10^?6M), by itself, had no effect on the FSH action. However, when added together with FSH and GnRH, antagonist A completely abolished the inhibitory effect of GnRH. These results demonstrate that the direct inhibitory effect of GnRH on granulosa cell function can be prevented by a GnRH antagonist and that the GnRH action at the ovarian level may require stringent stereospecific interactions of these peptides with putative GnRH recognition sites.     

Progesterone-induced inactivation of nuclear estrogen receptor in the hamster uterus is mediated by acid phosphatase

Inactivation of hamster uterine estrogen receptor was assayed in nuclear KCl extracts (30 min, 37°C, pH 7.5) after progesterone treatment $\text{in}\text{vivo}$ for 2h. At very low concentrations ($\text{>}$0.05 mM), molybdate and vanadate blocked the progesterone-induced increase in receptor inactivation. In contrast, only high concentrations ($\text{>}$10 mM) of the inhibitors blocked receptor inactivation in extracts from untreated hamsters. Gel electrophoresis and inhibition curves for phosphatases in nuclear extract demonstrated that acid, rather than alkaline, phosphatase activity is most likely responsible for these effects. These data suggest that progesterone antagonizes estrogen action in the hamster uterus by promoting estrogen receptor dephosphorylation leading to inactivation.     

Effects of the antioestrogen, ICI 164,384, on oestrogen induced RNAs in MCF-7 cells

The effects of ICI 164,384 on the expression of six oestrogen-regulated RNAs (pNR-1, pNR-2, pNR-13, pNR-17, pNR-25 and pNR-100) and the 46 kDa secreted protein were measured in MCF-7 cells. In marked contrast to tamoxifen, an antioestrogen commonly used in the treatment of breast cancer, ICI 164,384 administered alone had little or no effect on the RNAs or protein. ICI 164,384 completely inhibited the induction of the RNAs and 46 kDa protein by oestradiol. Although ICI 164,384 has an affinity for the human oestrogen receptor only slightly less than that of oestradiol, half maximal inhibition of oestradiol action was attained with between a 50 and 150-fold molar excess of ICI 164,384. The pNR-1 RNA is induced by tamoxifen but this induction was abolished by ICI 164,384. Thus, ICI 164,384 acts as a potent antioestrogen for the regulation of the expression of specific oestrogen-responsive genes in human breast cancer cells.     

Study of the in-vivo antioestrogenic action of N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine HCl (DPPE), a novel intracellular histamine antagonist and antioestrogen binding site ligand

N,N-Diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine HCl (DPPE) binds with high affinity to the antioestrogen binding site (AEBS), but not to the oestrogen receptor. There is an association of AEBS with a novel intracellular histamine receptor (H1C) of micromolar affinity through which histamine acts as a second messenger. An optimal dose of 4 mg DPPE/kg antagonized the uterine growth-stimulating effects of oestradiol in immature oophorectomized rats. Unlike tamoxifen, DPPE alone was not a partial agonist, but decreased uterine size and weight below control values at concentrations between 0.1 and 75 mg/kg. DPPE also antagonized oestradiol-stimulated uterine growth at 72 h; the inhibition observed was not significantly different from that seen with tamoxifen. Oestradiol-treated animals receiving the combination of DPPE (4 mg/kg) + low dose tamoxifen (0.04 mg/kg) for 72 h had significantly smaller uteri than did those receiving the same dose of DPPE or tamoxifen alone. Histologically, either DPPE or tamoxifen antagonized oestradiol stimulation of eosinophil migration and glandular epithelial proliferation; the latter inhibition was significantly greater for DPPE + tamoxifen (0.04 mg/kg) than for the same dose of DPPE or tamoxifen alone. Unlike tamoxifen, DPPE did not antagonize oestradiol stimulation of luminal epithelial proliferation, but in the presence of oestradiol, DPPE significantly decreased tamoxifen (0.65 mg/kg)-induced hypertrophy of the luminal epithelium. Based on these findings, we suggest that binding to the AEBS/intracellular histamine receptor is important to the action of antioestrogens.     

Effect of androgen mediated by the estrogen receptor of fish liver: Vitellogenin accumulation

We have investigated the action of high doses of androgens in $\text{Gobius niger L.}$, a marine teleostean fish, by characterizing specific steroid receptors in liver and by assaying the plasma vitellogenin concentration under different hormonal treatments. Estrogen and androgen receptors were characterized in the liver nuclear extracts according to their binding specificity. The maximum binding capacity was 25 fmoles/mg protein for the estrogen and androgen receptors. $\text{In vivo}$, high doses of DHT(·) increased the concentration of plasmatic vitellogenin as assayed by immunodiffusion while low doses were inefficient. In spite of a similar number of estrogen and androgen nuclear receptor sites (25 fmoles/mg protein), DHT was at least 70 fold less active than et on yolk protein and vitellogenin induction both in male and female $\text{Gobius niger}$. In addition, the antiestrogen tamoxifen, which was inactive by itself, inhibited the e₂ and the DHT induced accumulation of vitellogenin. Progesterone (2 mg/fish) was also totally inactive in inducing vitellogenin. We conclude that the induction of vitellogenin by DHT is mediated by the estrogen receptor rather than by the androgen receptor.In addition to the estradiol induced protein in rat uterus and to other estrogenic responses obtained by androgens in mammary cancer, fish vitellogenin is another estrogen regulated protein which can be induced by high doses of androgens. (·) 17β-hydroxy-5α-androstan-3-one.     

Effects of cholinergic drugs on growth hormone release

Acetylcholine and the cholinergic agonists, pilocarpine and physostigmine, increased GH release $\text{in}$$\text{vivo}$. The increase in GH release by pilocarpine was reversed by concurrent administration of the cholinergic receptor blocker, atropine, whereas atropine alone did not alter serum GH concentrations. Cholinergic stimulation of GH release appears to be partially mediated through a catecholaminergic system since the response was partially inhibited by pimozide, a dopamine receptor blocker, or phentolamine, an α-adrenergic receptor blocker. The cholinergic system may function physiologically to help regulate GH release.     

Noncholecystokinin peptides in human serum which cause gallbladder contraction

In fasting human serum, cholecystokinin (CCK) is not the principal substance which causes $\text{in}$$\text{vitro}$ rabbit gallbldder contraction. Removal of CCK by affinity chromatography from fasting sera from 8 healthy adults reduced bioactivity only by 18 ± 4% (SEM). Unlike CCK, the bioactivity of serum was enhanced by 30 to 57% rather than destroyed by pronase and chymotrypsin respectively and was not inhibited by dibutyryl cGMP. Reduction of serum bioactivity by carboxypeptidase Y indicated that the bioactive substances in serum are peptides. On Sephadex G-50, bioactive substances eluted in positions different from any known form of CCK. Thus, the principal substances in fasting human serum causing $\text{in}$$\text{vitro}$ gallbladder contraction are not CCK but are most likely small peptides which act at receptors different from the receptors for CCK.     

DDT inhibition of Ca-Mg ATPase from peripheral nerves and muscles of lobster, Homarus americanus

Sensitivity of CaMg ATPase from axonic plasma membrane (APM) and sarcoplasmic reticulum (SR) of lobster, $\text{Homarus}\text{,}\text{americanus}$, to DDT was studied. The CaMg ATPase found in SR with the high Ca²⁺ affinity is sensitive to DDT while the portion of ATPase related to the low Ca²⁺ affinity site is not inhibited by DDT. Also, DDT is more inhibitory against the CaMg ATPase prepared from APM than the one obtained from SR. The relationship between inhibition of the CaMg ATPase by DDT in the axonic nerve membrane and $\text{in}\text{,}\text{vivo}$ poisoning symptoms of the nervous system is discussed.     

Effect of morphine sulfate on adenylate cyclase and phosphodiesterase activities in rat corpus striatum.

The effect of morphine sulfate (MS) on adenylate cyclase (AC) and phosphodiesterase (PDE) activities in the rat striatum was investigated. MS produced a dose-dependent increase in basal AC activity and did not alter sodium fluoride-induced stimulation both $\text{in}$$\text{vivo}$ (7.5–30 mg/kg, 1 hr pretreatment, i.p.) and $\text{in}$$\text{vitro}$ (1–100μM). $\text{in}$$\text{vitro}$, when submaximal effective concentrations of dopamine and MS were combined, there was an additive effect. However, administration of MS $\text{in}$$\text{vivo}$ did not alter dopamine-induced stimulation of AC activity. MS, $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ inhibited PDE activity in a dose-dependent manner only with the high substrate concentration (3.3 × 10⁻³M cyclic AMP). Preliminary results from this study indicate that morphine affects the cyclic AMP system.     

Induction of ornithine decarboxylase in macrophages by bacterial lipopolysaccharides (LPS) and mycobacterial cell wall material

Lipopolysaccharide from $\text{E. Coli}$ (LPS) and BCG cell walls (BCGcw) are recognized immunoadjuvants that directly stimulate some macrophage functions. The macrophage cell line J774.1 and peritoneal exudate cells (PEC) from mice can be stimulated by LPS or other adjuvants $\text{in vitro}$ to synthesize and release protein factor(s) that activate thymus-derived lymphocytes. We have utilized J774.1 cells and PEC to demonstrate that an increase in ornithine decarboxylase (ODC) activity is a marker of early biochemical changes in adjuvant-stimulated macrophages. BCGcw and LPS increased ODC within 2 hours in J774.1 cells as well as murine peritoneal exudate macrophages. Maximal increases in ODC were detected 4 hours after the addition of adjuvants to J774.1 cells. The marked increases (12–23 fold) in ODC observed with BCGcw (20 μg/ml) did not appear to involve an effect on cell proliferation which was suppressed by this adjuvant. Cycloheximide inhibited the induction of ODC by LPS and BCGcw in the macrophage cell line. Evidence that the induction of ODC may be promoted by an increase in cyclic AMP was provided by experiments demonstrating that prostaglandin E₁ (PGE₁) and 8-bromo-adenosine-3′:5′-monophosphate (8Br-cyclic AMP) can mimic the effects of LPS and BCGcw in J774.1 cells. These observations indicate that one of the early biochemical changes in macrophages promoted by adjuvants is an induction of ODC.     

The effects of quipazine on serotonin metabolism in rat brain.

Quipazine, 2-(1-piperazinyl)-quinoline, is a drug that has been reported to stimulate serotonin receptors in brain. We therefore studied the effect of quipazine on several parameters of serotonin metabolism in rat brain. Quipazine caused a slight, dose-related elevation of serotonin levels and decrease in 5-hydroxyindoleacetic acid levels for 2–4 hrs after it was administered. The decrease in 5-hydroxyindoleacetic acid levels was probably due primarily to a depression of 5-hydroxyindole synthesis, since quipazine also decreased the rate of 5-hydroxytryptophan accumulation after NSD 1015, the rate of serotonin decline after α-propyldopacetamide, and the rate of 5-hydroxyindoleacetic acid accumulation after probenecid. The elevation of serotonin was probably due to weak inhibition of monoamine oxidase. Quipazine reversibly inhibited the oxidation of serotonin by rat brain monoamine oxidase $\text{in}$$\text{vitro}$ and protected against the irreversible inactivation of the enzyme $\text{in}$$\text{vivo}$. Quipazine also was a potent inhibitor of serotonin uptake into brain synaptosomes $\text{in}$$\text{vitro}$ and attained concentrations in brain higher than the $\text{in}$$\text{vitro}$ IC₅₀. However, quipazine did not prevent the depletion of brain serotonin by p-chloroamphetamine $\text{in}$$\text{vivo}$. In addition to stimulating serotonin receptors in brain, quipazine may inhibit monoamine oxidase and serotonin reuptake $\text{in}$$\text{vivo}$.     

Danazol inhibits human adrenal 21-and 11β-hydroxylation invitro

The effects of danazol on steroidogenesis $\text{in}$$\text{vitro}$ in the 16–20 week old human fetal adrenal were examined by studying: 1) danazol binding to adrenal microsomal and mitochondrial cytochrome P-450, and 2) enzyme kinetics of danazol inhibition of the adrenal microsomal 21-hydroxylase and the mitochondrial llβ-hydroxylase. The addition of danazol to preparations of adrenal microsomes or mitochondria elicited a type I cytochrome P-450 binding spectrum. Danazol bound to microsomal cytochrome P-450 with a high affinity apparent spectral dissociation constant (Kg) of 1 μM and with a lower affinity K's of 10 μM. Danazol bound to mitochondrial cytochrome P-450 with a Kg of 5 μM. In addition, danazol competitively inhibited the microsomal 21-hydroxylase (apparent enzymatic inhibition constant K_I = 0.8 μM) and the mitochondrial 11β-hydroxylase (K_I = 3 μM). These findings demonstrate that low concentrations of danazol directly inhibit steroidogenesis in the human fetal adrenal $\text{in}$$\text{vitro}$.     

Dibutyryl cGMP: inhibitor of the effect of cholecystokinin and gastrin on the guinea pig gallbladder in vitro

N², O²-di-butyryl guanosine 3′:5′ monophosphate (Bt₂ cGMP), a known competitive and selective inhibitor of the effect of cholecystokinin on the pancreatic acinar cells $\text{in}$$\text{vitro}$ was tested for its effect on the guinea pig gallbladder $\text{in}$$\text{vitro}$. Bt₂ cGMP inhibited competitively the contractile effect of cholecystokinin octapeptide, and also inhibited the contraction induced by sulfated gastrin-17. Bt₂ cGMP failed to inhibit the contraction induced by bombesin, acetylcholine or histamine. The 8-bromo derivative of cGMP and the dibutyryl derivative of cAMP did not affect contraction stimulated by cholecystokinin octapeptide. Since it is specific for gastrincholecystokinin peptides, and not restricted to the pancreas, Bt₂ cGMP could be used to recognize the action of these peptides.     

Resistance of Tetrahymena to ricin, a toxic enzyme from Ricinus communis.

The protozoan $\text{Tetrahymena}\text{pyriformis}$ was found to be resistant to the toxic action of ricin $\text{in}\text{vivo}$. Isolated $\text{Tetrahymena}$ ribosomes were strongly resistant to the A subunit of ricin when tested in a cell free protein synthesis system under different conditions and also lacked the ability to bind A chain stoichometrically. This suggests that $\text{Tetrahymena}$ is resistant $\text{in}\text{vivo}$ because it contains a ribosome which is not susceptible to the toxic action of ricin.     

Specificity of gentamicin accumulation by rat renal cortex.

The accumulation of gentamicin by rat renal cortex $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$ was not inhibited by probenecid, tetraethylammonium, cephalosporins nor α-aminoisobutyric acid, but was significantly blocked by other aminoglycosides (neomycin, tobramycin and kanamycin). The data suggest that specific binding sites for aminoglycosides are present on the surface or in cells of the renal proximal tubule.     

Enkephalin analogues and naloxone modulate the release of growth hormone and prolactin - evidence for regulation by an endogenous opioid peptide in brain

Met⁵-enkephalin amide, D-Ala²-Met⁵-enkephalin amide, D-Ala²-Leu⁵-enkephalin amide, morphine sulfate and naloxone hydrochloride were examined for their effects on growth hormone and prolactin release $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. Subcutaneous injection of D-Ala²-Met⁵ enkephalin amide^a, D-Ala²-Leu⁵ enkephalin amide^b and morphine sulfate, but not Met⁵-enkephalin and amide^c, resulted in significant elevations in the serum growth hormone and prolactin of immature female rats. Naloxone blocked the hormone-stimulatory effect of the opioid receptor agonists and when administered alone significantly reduced serum growth hormone and prolactin concentrations. None of the drugs demonstrated a direct action on anterior pituitary tissue growth hormone or prolactin release $\text{in}$$\text{vitro}$.     

Comparison in male and female rats of ACTH content and response to corticotrophin-releasing factor (CRF) of cultured adenohypophyseal cells and in vivo hypothalamic CRF content.

Although mean ACTH concentration was significantly higher in cultured adenohypophyseal cells from female than from male rats, there was no significant sex difference in the ACTH secretory response of these cells to hypothalamic extract containing CRF. Hypothalamic CRF content in the “basal” state $\text{in}$$\text{vivo}$ was slightly higher in female than $\text{in}$ male rats.     

Serotonin action on short-circuit current and ion transport across isolated rabbit ileal mucosa.

Serotonin has previously been implicated as the cause of the diarrhea associated with carcinoid syndrome and the amine has been shown by others to be an intestinal secretagogue in preparations of intestinal loops $\text{in}$$\text{vivo}$. In the present paper the action of serotonin on isolated segments of rabbit ileal mucosa stripped of muscle layers was studied $\text{in}$$\text{vitro}$. Serotonin (10^?4M) caused an abrupt significant rise in short-circuit current (Isc) across the mucosal epithelial cell layer but this effect was transient. No change was observed in tissue conductance. In this preparation, serotonin did not alter ²²Na, ³⁶Cl or residual ion fluxes across the mucosa. High blood serotonin levels for a period of several days also did not alter ion fluxes or Isc in isolated rabbit ileum. Therefore, it is concluded that serotonin must cause its secretory activity observed $\text{in}$$\text{vivo}$ by some mechanism other than a direct action on epithelial cell transport mechanisms.