A dicarba analog of beta-atrial natriuretic peptide (beta-ANP) inhibits guanosine 3',5'-cyclic monophosphate production induced by alpha-ANP in cultured rat vascular smooth muscle cells

The synthesis and biological properties are described of [Asu7,23']-beta-ANP-(7-28) (Asu, L-alpha-aminosuberic acid), a dicarba analog of beta-atrial natriuretic peptide (beta-ANP, an antiparallel dimer of human alpha-ANP with the chains linked by 7-23' and 7'-23 disulfide bonds). This Asu-analog (referred to as analog III) displaced 125I-alpha-ANP specifically bound to cultured rat vascular smooth muscle cells (VSMC) with an apparent Ki of 2.1 x 10(-8) M, but did not stimulate formation of intracellular cGMP at 10(-8) -10(-5) M. Analog III inhibited the alpha-ANP-stimulated cGMP production in VSMC competitively with a pA2 value of 7.45 and behaved as an antagonist of alpha-ANP in rat aorta smooth muscle relaxation. In addition, beta-ANP was also shown to inhibit the alpha-ANP-induced cGMP production in a dose-dependent manner. The mechanism of action of beta-ANP is also discussed.     

The inhibition of progesterone secretion and the regulation of cyclic nucleotides by atrial natriuretic factor in gonadotropin responsive murine Leydig tumor cells

We have found that atrial natriuretic factor (ANF) has a profound effect on testicular cells in altering intracellular cyclic nucleotide levels as well as progesterone secretion. Using clonal cultured Leydig tumor cells we found that 1 X 10(-8)M ANF caused a two thousand-fold elevation in the accumulation of cellular cGMP and inhibited cAMP in treated cells by more than 90% as compared to the controls. ANF (1 X 10(-8)M) also significantly inhibited gonadotropin-stimulated accumulation of cAMP in response to bovine luteinizing hormone (bLH) or human chorionic gonadotropin (hCG). Gonadotropin-stimulated progesterone secretion was inhibited by ANF (1 X 10(-10) - 1 X 10(-9)M) in these cultured Leydig tumor cells. Approximately 50% inhibition of progesterone secretion was observed at the peptide concentration of 1 X 10(-9) M.     

Total and free testosterone in plasma of hypo- and agonadal men.

Plasma total testosterone (T), apparently free T and testosterone binding globulin (TeBG) capacity determined in 14 normal men aged 30-40 years were 461 +/- 100 ng/100 ml, 9.4 +/- 3.0 ng/100 ml and 5.7 +/- 1.9 X 10(-8) M, respectively, whereas in 16 hypogonadal men the corresponding values were 38.6 +/- 27.2 ng/100 ml, 0.47 +/- 0.41 ng/100 ml and 10.4 +/- 3.4 X 10(-8) M showing the TeBG capacity significantly higher (p less than 0.001) in hypogonadal than in normal men. Treatment of 5 hypogonadal subjects with 250 mg testosterone enanthate plus 50 mg testosterone propionate decreased (p less than 0.001) the TeBG level from 14.7 +/- 2.5 X 10(-8YM to 8.3 +/- 1.4 X 10(-8) M on day 8 after a single injection. According to this difference in TeBG, the free T fraction in plasma rose from 0.94% to 1.9% of the total T concentration. These results suggest that alteration of total plasma T affected the TeBG capacity. Decreased T levels raised and increased T concentrations suppressed TeBG, but with a delayed response to the changed T concentrations. The initial mean values in 12 patients with prostatic cancer aged 60-74 years were 397 +/- 165 ng/100 ml, 4.05 +/- 1.8 ng/100 ml and 11.9 +/- 3.3 X 10(-8) M, respectively. The TeBG capacity in these patients was significantly higher and the free T concentration significantly lower (p less than 0.001) than those of the younger normal males. After treatment with 12 g diethylstilbestrol diphosphate and orchidectomy, the TeBG increased to 33.3 +/- 13.1 X 10(-8) M and the plasma free T concentration decreased to the minimal value of 0.053 +/- 0.04 ng/100 ml.     

Effect of atrial natriuretic peptide on arterial pressure and renal function in cirrhotic rats with ascites

Glomerular filtration rate, urine volume, sodium excretion and mean arterial pressure were measured in 10 rats with Cl4C induced cirrhosis presenting sodium retention and ascites, and in 10 control rats before and during the iv administration of the 28 aminoacid rat alpha-Atrial Natriuretic Peptide (alpha-ANP) (a bolus of 1 microgram followed by a constant infusion of 33 ng/min). alpha-ANP induced a similar increase in glomerular filtration rate and filtered sodium load in both groups of rats. In contrast, the increase in urine volume and sodium excretion produced by alpha-ANP was significantly lower in cirrhotic rats (from 13.8 +/- 1.9 to 37.9 +/- 9.1 microliters/min., and from 0.5 +/- 0.1 to 3.3 +/- 1.0 microEq/min) than in control animals (from 14.6 +/- 1.3 to 102.5 +/- 17.7 microliters/min., p less than 0.005; and from 1.0 +/- 0.3 to 14.1 +/- 3.2 microEq/min., p less than 0.001). The results indicate that in rats with experimental cirrhosis and ascites there are blunted diuretic and natriuretic responses to alpha-ANP, probably as a consequence of the exaggerated tubular sodium reabsorption present in these animals.     

Comparison of detecting sensitivities of different sizes of gold particles with electron-microscopic immunogold staining using atrial natriuretic peptide in rat atria as a model

The detecting sensitivities of different-sized gold particles were compared in the localization of atrial natriuretic peptide (ANP) in rat atria. The secondary antibodies were goat antirabbit labeled with 5, 15, 30, or 40 nm colloidal gold diluted 1:2 to 1:100 in Tris buffer. The relative quantity of alpha-ANP immunoreactivity in specific granules was determined by subtracting the number of gold particles in 1 micron 2 nongranule area from that in 1 micron 2 granule area measured with a computerized image analyzer. The optimal dilution that achieved the maximal contrast between specific and background label was influenced by the particle size. Optimal dilutions were 1:80, 1:30, 1:20, and 1:5 for 5, 15, 30, and 40 nm gold, respectively. At optimal dilutions, the maximal detecting sensitivity (MDS) was in inverse proportion to the gold particle size; however, this relationship is not entirely linear. The ratio among the MDSs of 5, 15, 30, and 40 nm gold particles was approximately 34:9:3:2. A double immunogold staining was performed to localize alpha- and beta-ANPs with 15 and 5 nm gold, respectively. Both antigens were detected in the same granules. If the ratios established from the single staining data were used, the ratio between the alpha- and the beta-ANP antigens in the same granules was approximately 2.8:1. The data obtained in this study provide a useful reference for applications of immunogold electron microscopy in a quantitative manner, particularly for double immunogold labeling.     

Effects of atrial natriuretic factor on human platelet function

We examined the hypothesis that atrial natriuretic factor (ANF), a substance with known vasorelaxant activities, shares with other vasodilators the property of inhibiting platelet function. Aggregation of citrated platelet-rich plasma (PRP) from 23 healthy volunteers induced by ADP, adrenaline, arachidonic acid, collagen, gamma-thrombin, the endoperoxide analogue U-44069, serotonin, the calcium ionophore A-23187 or platelet aggregating factor was measured after incubation of PRP with ANF for 3 minutes at concentrations of 4 X 10(-9), 4 X 10(-8) and 4 X 10(-7) M or vehicle as control. ANF decreased ADP-induced aggregation significantly (P less than 0.02), but only at the highest concentration used and to a minor extent (control: 73.6 +/- 11.2%; after ANF 4 X 10(-7) M: 60.0 +/- 17.1%, mean +/- S.D., n = 39) by a selective inhibitory effect on the secondary wave; neither aggregation by all other agents tested nor thromboxane B2 generation induced by ADP and adrenaline was altered by incubation with ANF. Although ANF thus has detectable effects on ADP-induced platelet aggregation in vitro, these data suggest that ANF is unlikely to be a physiologically significant modulator of platelet function.     

Steroidogenic effect of atrial natriuretic factor in isolated mouse Leydig cells is mediated by cyclic GMP.

The effects of different atrial natriuretic peptides on cyclic GMP formation and steroidogenesis have been studied in Percoll-purified mouse Leydig cells. Rat atrial peptides rANP (rat atrial natriuretic peptide), rAP-I (rat atriopeptin I) and rAP-II (rat atriopeptin II), in the presence of a phosphodiesterase inhibitor, stimulated cyclic GMP formation in a concentration-dependent manner. In the presence of saturating concentrations of the peptides, a 400-600 fold stimulation of cyclic GMP accumulation was observed. Among the peptides, rAP-II appeared to be the most potent. ED50 values (concentration causing half-maximal effect) for rAP-II, rANP and rAP-I were 1 X 10(-9) M, 2 X 10(-9) M and 2 X 10(-8) M respectively. A parallel stimulation of cyclic GMP formation and testosterone production by the cells was observed after incubation of the cells with various concentrations of rAP-II. In the presence of a saturating concentration of rAP-II (2 X 10(-8) M), maximum stimulation of intracellular cyclic GMP content was obtained within 5 min of incubation. Testosterone production by mouse Leydig cells could be stimulated by 8-bromo cyclic GMP in a concentration-related manner. At a 10 mM concentration of the cyclic nucleotide, steroidogenesis was stimulated to a similar extent as that obtained with a saturating concentration of human chorionic gonadotrophin (5 ng/ml). On the basis of these results we conclude that cyclic GMP acts as a second messenger in atrial-peptide-stimulated steroidogenesis in mouse Leydig cells. The steroidogenic effect of atrial peptides appears to be species-specific, since none of these peptides stimulated testosterone production by purified Leydig cells of rats, though in these cells a 40-60-fold stimulation of cyclic GMP formation in response to each of the three peptides was observed. However, 8-bromo cyclic GMP could stimulate testosterone production in rat Leydig cells. Therefore we conclude that the lack of steroidogenic response in rat Leydig cells to atrial-natriuretic-factor-stimulation results from an insufficient formation of cyclic GMP in these cells. This species difference would appear to result from a lower guanylate cyclase activity in rat Leydig cells.     

Inhibition of mast cell chymase by eglin c and antileukoprotease (HUSI-I). Indications for potential biological functions of these inhibitors

Recombinant eglin c (originally isolated from the medical leech) and antileukoprotease (HUSI-I from human seminal plasma) were examined for their ability to inhibit the mastcell protease chymase. Both inhibitors react rapidly with the enzyme: when about equimolar concentrations (in the range of 10(-8) M) of chymase and HUSI-I or eglin c were incubated the complex formation was apparently at equilibrium after 1 or 5 min respectively. When a constant amount of chymase (approximately 3 X 10(-8) M) was incubated with increasing concentrations of inhibitor a concentration of HUSI-I of 7 X 10(-7) M was necessary to cause 50% inhibition of the initial enzyme activity, whereas 8 X 10(-8) M eglin c was sufficient. The dissociation constant of the chymase-eglin c complex was calculated to be 4.4 X 10(-8) M. These results are discussed with respect to the possible in vivo function of antileukoprotease as an inhibitor of mast cell chymase.     

Enkephalinergic control of somatostatin secretion from the perfused rat stomach

A role for the enkephalins in the regulation of gastric somatostatin (SLI) secretion has been investigated in an isolated perfused rat stomach model. Both methionine- and leucine-enkephalins caused a dose-dependent inhibition of gastric inhibitory polypeptide (GIP) stimulated SLI secretion. Leu-enkephalin was one order of magnitude less potent than met-enkephalin: 50% inhibition by met-enkephalin was at 4 X 10(-9) M and with leu-enkephalin 3.5 X 10(-8) M. Naloxone (100 nM) had no effect on basal secretion but blocked the inhibitory action of met-enkephalin (1 nM or 1 microM). Vagal stimulation (7 V, 10 Hz, 5 ms) inhibited GIP-stimulated SLI release. Administration of naloxone partially reversed this inhibition, suggesting that endogenous opioids were at least partially responsible for vagally induced inhibition. A number of possible pathways by which endogenous enkephalins may modulate SLI release have been proposed.     

The effect of prostaglandins E1, E2, F1 alpha and F2 alpha on pig erythrocytes during haemolysis induced with aspirin and hypotonic NaCl solution

The effect of prostaglandins PGE1, PGE2, PGF1 alpha and PGF2 alpha was investigated on the haemolysis of pig erythrocytes induced with aspirin and hypotonic (0.119 M) NaCl solution. An inhibiting effect was observed of low concentrations (2 X 10(-5) M, 2 X 10(-4) M and 2 X 10(-3) M) of aspirin on haemolysis induced with hypotonic NaCl solution, while in a concentration of 2 X 10(-2) M aspirin itself caused haemolysis which amounted to 93% of the haemolysis induced with 0.041 M NaCl solution. No differences were observed in the degree of haemolysis inhibition in relation to the time of incubation of erythrocytes with aspirin. Aspirin concentrations from 0.035 M to 0.280 M caused slight haemolysis (9-15% of the haemolysis induced with water), the 0.560 M solution caused haemolysis corresponding to 85% of the water-induced haemolysis. None of the studied prostaglandins used in concentrations of 0.4 X 10(-3) M, 0.4 X 10(-4) M and 0.4 X 10(-5) M had any significant effect on aspirin-induced haemolysis. PGE1 and PGE2 in concentrations of 0.4 X 10(-3) M, 0.4 X 10(-4) M and 0.4 X 10(-5) M inhibited haemolysis induced with 0.119 M sodium chloride solution, and the degree of haemolysis inhibition was from 8% to 35%. Prostaglandins PGF1 alpha and PGF2 alpha in the same concentrations had no protective effect.     

Prostaglandin inhibition of testosterone production induced by luteinizing hormone, dibutyryl cyclic AMP or 3-isobutyl-1-methyl xanthine in dispersed rat testicular interstitial cells.

Testicular interstitial cells were utilized to study the effects of prostaglandins (PG) on in vitro testosterone production and to examine the role of cyclic adenosine-3',5'-monophosphate (cAMP) in this process. Testosterone production was assessed after 3 hour incubations while cAMP accumulation was examined after a 0.5 hour incubation period. Testosterone and cAMP were measured by radioimmunoassay. None of the PGs tested (PGA, PGA2, PGB1, PGE1, PGE2, PGF1alpha PGF2alpha) altered basal testosterone production when present in incubates at concentrations of 1.3 X 10(-8) M to 1.3 X 10(-4). However, at concentrations of 1.3 X 10(-4) M all of these PGs were capable of decreasing Luteinizing Hormone (LH; 100ng)-induced testosterone production. The inhibition of LH-induced testosterone production by the B, E and F series PGs was less pronounced than that for the A series. PGA1 and PGA2 exhibited 80% and 95% inhibition, respectively, at 1.3 X 10(4) M. The inhibitory action of 4 X 10(5) M PGA1 or PGA2 was evident within 30 minutes. Preincubation of interstitial cells with indomethacin (10(-5) or 10(-6) M) for 30 minutes did not alter subsequent basal or LH (100ng)-induced testosterone production. Accumulation of cAMP was stimulated by LH (10 microgram) or by PGs (1.3 X 10(-4) M PGA1, PGA2, PGB1, PGE1 or PGF2alpha). The PG-induced cAMP accumulation thus occurred at concentrations where LH-stimulated testosterone production was inhibited. Furthermore, PGA1 and PGA2 (1.3 X 10(-4) M) inhibited testosterone production induced by either 3-isobutyl-1-methyl xanthine (MIX; 10(-4) M or 10(-3) M) or dibutyryl cAMP (dbcAMP; 10(-4) M or 10(-3) M). These results indicate that PGs can block testosterone production by a direct effect on testicular interstitial cells and suggest that PGs exert their inhibitory action distal to stimulation of cAMP formation. PGs do not appear to play a role in the mechanism of LH action.     

Effect of serotonin on murine macrophages: suppression of Ia expression by serotonin and its reversal by 5-HT2 serotonergic receptor antagonists

Serotonin (5-HT), a mediator released from platelets at sites of inflammation, suppressed IFN-gamma-induced Ia expression in mouse bone marrow macrophages maintained in vitro. (Mean percent suppression = 63.9% +/- 9.2, n = 40.) This suppression was not toxic or endotoxin-related, was concentration-dependent, and occurred at the physiologic concentrations of 5-HT present at inflammatory sites. The concentration of 5-HT producing the half-maximal effect was 2.5 to 5.5 X 10(-8) M. Related compounds, dopamine, histamine, and tryptamine, were much less potent in suppressing IFN-gamma-induced Ia, with maximally suppressing concentrations more than 100-fold higher than the maximally suppressing 5-HT concentration. L-5-hydroxytryptophan (5-HTP), the most potent analog tested, was 10-fold less potent than 5-HT in suppressing Ia expression. The concentration of 5-HTP producing the half-maximal effect = 4 X 10(-7) M. 5-HT suppression of IFN-gamma-induced Ia expression was antagonized by the 5-HT2 type receptor antagonists spiperone, ketanserin, and LY53857. Concentrations of these agents resulting in 50% inhibition of the serotonin effect were 1.5 X 10(-8) M, 7.5 X 10(-8) M, and 4.5 X 10(-12) M, respectively. 5-HT was most effective in suppressing IFN-gamma-induced Ia when added early in culture simultaneously with IFN-gamma. These data provide functional evidence that 5-HT suppression of IFN-gamma-induced Ia expression is mediated through a 5-HT receptor with some characteristics of the 5-HT2 type. 5-HT may play a physiologic role at sites of inflammation as a modulator of the effects of IFN-gamma on macrophage function.     

Ca2+ Regulates Hormone Secretion and Proopiomelanocortin Gene Expression in Melanotrope Cells via the Calmodulin and the Protein Kinase C Pathways

The mechanism by which Ca2+ regulates proopiomelanocortin (POMC)-derived peptide secretion and POMC mRNA levels was investigated in primary cultures of porcine intermediate lobe (IL) cells maintained in serum-free medium. POMC gene expression was evaluated by the dot blot hybridization assay with a 32P-labeled DNA probe complementary to the full-length sequence of porcine POMC mRNA. Treatment of IL cells for 24 h with the calmodulin (CAM) antagonists W7 and W13 reduced POMC mRNA levels by a maximum of 50% in a dose-dependent manner (ED50 approximately 10(-8) M). Accumulation of alpha-melanocyte-stimulating hormone (alpha-MSH) in the medium was also depressed by 50% after 8 h of treatment. The role of protein kinase C (PKC) was investigated by depleting the IL cell PKC content with phorbol ester treatment. Phorbol 12-myristate 13-acetate (PMA) at 5 X 10(-8) M induced a rapid translocation of cytoplasmic PKC activity toward the membrane. After 12 h of PMA treatment, PKC activity was undetectable in either the cytoplasmic or the particulate fractions. The same dose of PMA induced a time-dependent decrease in POMC mRNA levels (50% inhibition after 24 h). The same effect was seen with the phorbol ester phorbol 12,13-dibutyrate at 5 X 10(-8) M, whereas the inactive phorbol ester 4 alpha-phorbol at 5 X 10(-8) M was without effect after 24 h of treatment. PMA treatment had a biphasic effect on alpha-MSH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Existence of endogenous inhibitors of platelet-activating factor (PAF) with PAF in rat uterus

Two kinds of phospholipids in normal rat uterus were found to inhibit the aggregation of washed rabbit platelets induced by 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC) and were named Inhibitor I and Inhibitor II and identified by mass spectrometry. Inhibitor I was a mixture of 1-acyl (16:0, 18:0, 18:1, 18:2, and 20:4)-2-lyso-sn-glycero-3-phosphocholine (acyllyso-GPC) and 1-alkyl (16:0, 18:0, and 18:1)-2-lyso-sn-glycero-3-phosphocholine (alkyllyso-GPC). 16:0 acyllyso-GPC was the most inhibitory, followed by 18:1, 18:2, 20:4, and 18:0 acyllyso-GPCs and 16:0 alkyllyso-GPC. Their IC50 values were in the range of 1-4 X 10(-5) M against the platelet aggregation induced by 1 X 10(-10) M 16:0 alkylacetyl-GPC, indicating that they were about 100 times weaker inhibitors than CV-3988. Inhibitor II was a mixture of N-acyl sphing-4-enyl phosphocholine (18:1/18:0, 18:1/20:0, 18:1/24:0, and 18:1/24:2). The most inhibitory of these components were 18:1/20:0 and 18:1/24:0, followed by 18:1/24:2 and 18:1/18:0, and their IC50 values were in the range of 4-5 X 10(-5) M against platelet aggregation induced by the alkylacetyl-GPC. Quantitatively, about 10(5) times higher concentrations of these inhibitors should be necessary to inhibit platelet aggregation induced by 1 X 10(-10) M 16:0 alkylacetyl-GPC. In fact, the contents of Inhibitors I and II, respectively, were approximately 10(5) times (4.7 X 10(-2) and 7.1 X 10(-2) mol/mol lipid-phosphorus of the original uterine phospholipids) than that of 16:0 alkylacetyl-GPC (1.4 X 10(-6) mol/mol lipid-phosphorus). The role of alkylacetyl-GPC in normal rat uterus is uncertain, but it coexists in situ with two kinds of endogenous inhibitors, choline containing lysoglycerophospholipids and sphingophospholipids.     

Differences among primates in defence against infection: sensitivity of polymorphonuclear leukocytes to fMet-Leu-Phe

The sensitivity of polymorphonuclear leukocytes (PMN) to N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) for chemotaxis and for lysosomal enzyme release was examined using the PMN of four primate species, human (H. sapiens), chimpanzee (P. troglodytes), rhesus monkey (M. mulatta), and cotton-headed tamarin (S. (O) oedipus). The 50 per cent effective concentrations (EC50) of fMet-Leu-Phe for chemotaxis were 2.5 X 10(-9) M in human, 10(-9) M in chimpanzee, 8 X 10(-8) M in rhesus monkey, and 3.3 X 10(-6) M in tamarin. The EC50 values of fMet-Leu-Phe for myeloperoxidase (MPO) release were 10(-8) M in human, 4 X 10(-8) M in chimpanzee, 4 X 10(-8) M in rhesus monkey, and 10(-6) M in tamarin and those for beta-glucuronidase release were 4 X 10(-9) M, 6.4 X 10(-8) M, 1.8 X 10(-7) M, and 1.6 X 10(-6) M, respectively. Thus, the sensitivity to fMet-Leu-Phe for chemotaxis was in the order: chimpanzee congruent to human greater than rhesus monkey greater than tamarin, and that for the release of lysosomal enzymes, MPO and beta-glucuronidase, was in the order: human greater than chimpanzee greater than rhesus monkey greater than tamarin. These results appear to indicate that the sensitivity to fMet-Leu-Phe increases in the order of evolution of primates toward the human, and suggest that the sensitivity of PMN in the defence function against infection also increases in the same order.     

Hormonal regulation of prolactin release by human prolactinoma cells cultured in serum-free conditions

Thyrotropin-releasing hormone (TRH) stimulates the prolactin (PRL) release from normal lactotrophs or tumoral cell line GH3. This effect is not observed in many patients with PRL-secreting tumors. We examined in vitro the PRL response to TRH on cultured human PRL-secreting tumor cells (n = 10) maintained on an extracellular matrix in a minimum medium (DME + insulin, transferrin, selenium). Addition of 10(-8) M TRH to 4 X 10(4) cells produced either no stimulation of PRL release (n = 6) or a mild PRL rise of 32 +/- (SE) 11% (n = 4) when measured 1, 2 and 24 h after TRH addition. When tumor cells were preincubated for 24 h with 5 X 10(-11) M bromocriptine, a 47 +/- 4% inhibition of PRL release was obtained. When TRH (10(-8) M) was added, 24 h after bromocriptine, it produced a 85 +/- 25% increase of PRL release (n = 8). This stimulation of PRL release was evident when measured 1 h after TRH addition and persisted for 48 h. The half maximal stimulatory effect of TRH was 2 X 10(-10) M and the maximal effect was achieved at 10(-9) M TRH. When tumor cells were pretreated with various concentrations of triiodothyronine (T3), the PRL release was inhibited by 50% with 5 X 10(-11) M T3 and by 80% with 10(-9) M T3. Successive addition of TRH (10(-8) M) was unable to stimulate PRL release at any concentration of T3. The addition of 10(-8) M estradiol for up to 16 days either stimulated or had no effect upon the PRL basal release according to the cases. In all cases tested (n = 4), preincubation of the tumor cells with estradiol (10(-8) M) modified the inhibition of PRL release induced by bromocriptine with a half-inhibitory concentration displaced from 3 X 10(-11) M (control) to 3 X 10(-10) M (estradiol). These data demonstrate that the absence of TRH effect observed in some human prolactinomas is not linked to the absence of TRH receptor in such tumor cells. TRH responsiveness is always restored in the presence of dopamine (DA) at appropriate concentration. This TRH/DA interaction seems specific while not observed under T3 inhibition of PRL. Furthermore, estrogens, while presenting a variable stimulatory effect upon basal PRL, antagonize the dopaminergic inhibition of PRL release.     

Inhibition of androgen and oestrogen production by clomiphene citrate in avian theca cells

Isolated theca cells (2 X 10(5)/ml) were pre-incubated for 1 h in the presence or absence of clomiphene citrate (10(-12)-10(-4) M). Ovine LH (50 ng/ml) was added and cells were incubated for an additional 3 h. A 50% inhibition of LH-stimulated androstenedione and oestrogen production was obtained with doses of 10(-8) M and 2 X 10(-7) M clomiphene, respectively. Furthermore, the effect of clomiphene on LH-stimulated androstenedione production was reversed by washing clomiphene from the cells before stimulation with LH. In subsequent experiments, the effects of clomiphene on C17-20-lyase and aromatase activities were examined. Conversion of [3H]17-hydroxyprogesterone to androstenedione was inhibited by 50% when theca cells were pretreated with 10(-5) M-clomiphene. In addition, conversion of testosterone to oestrogen by theca cells was inhibited in a dose-dependent manner by clomiphene, with 50% inhibition occurring at a dose of 5 X 10(-6) M. The results show that clomiphene treatment in vitro inhibits androgen and oestrogen production in theca cells by inhibitory effects on the activities of C17-20-lyase and aromatase. In addition to the widely-accepted effects of clomiphene on the hypothalamic-pituitary axis, the present findings add further support to the suggestion that clomiphene exerts direct effects on ovarian steroidogenesis.     

The muscarinic receptor of chick embryo cells: correlation between ligand binding and calcium mobilization

In this report we characterize muscarinic cholinergic receptor on embryonic cells. We established dose-response curves by fluorometric measurement of Ca2+ mobilization in cell suspensions of whole chick embryos stage 23/24. Ca2+ mobilization was quantitated by standardization of chlorotetracycline (CTC) fluorescence changes after stimulation with muscarinic agonists. We determined ED50 values for the agonists acetylcholine and carbachol as 3.4 X 10(-6) and 2.7 X 10(-5) M, respectively. Pilocarpine and oxotremorine were found to act as reversible competitive antagonists with inhibition constants (Kl) of 5.0 X 10(-6) and 1.4 X 10(-6) M, respectively. Bethanechol, which induced only 23% of the maximal effect obtained by acetylcholine, was a partial agonist with an ED50 of 4.8 X 10(-4) M. Its antagonistic component is expressed by an inhibition constant of 1.9 X 10(-4) M. In parallel, binding studies were performed in a competition assay with [3H]-quinuclidinylbenzilate. For the agonists acetylcholine and carbachol, binding parameters were best fitted by a "two binding-sites model." Comparison with dose-response curves indicated that Ca2+ mobilization was triggered via the high-affinity binding site. The inhibition constants of antagonists derived from the shift of dose-response curves corresponded to the fitted KD values of the binding studies when a "one binding-site model" was applied. Combination of dose-response and binding data showed close proportionality between receptor occupancy and calcium mobilization. No spare receptors were present.     

Prolonged exercise following diuretic-induced hypohydration effects on fluid and electrolyte hormones.

To investigate the hypothesis that a reduction in plasma volume (PV) induced by diuretic administration would result in an increase in the fluid and electrolyte hormonal response to exercise, ten untrained males (VO(2) peak = 3.96 +/- 0.14 l/min) performed 60 min of cycle ergometry at 61 % VO(2) peak twice. The test was carried out once under control conditions (CON) (placebo) and once after 4 days of diuretic administration (DIU) (Novotriamazide; 100 mg triamterene and 50 mg hydrochlorothiazide). Calculated resting PV decreased by 14.6 +/- 3.3 % (p < 0.05) with DIU. No difference in plasma osmolality was observed between conditions. For the hormones measured, differences (p < 0.05) between conditions at rest were noted for plasma renin activity (PRA) (0.62 +/- 0.09 vs. 5.61 +/- 0.94 ng/ml/h), angiotensin I (ANG 1) (0.26 +/- 0.03 vs. 0.56 +/- 0.08 ng/ml), aldosterone (ALD) (143 +/- 14 vs. 1603 +/- 302 pg/ml), arginine vasopressin (AVP) (4.13 +/- 1.1 vs. 9.58 +/- 1.6 pg/ml) and atrial natriuretic peptide (alpha-ANP) (11.5 +/- 2.8 vs. 6.33 +/- 1.0 pg/ml). The exercise resulted in increases (p < 0.05) in PRA, ANG I, ALD, AVP, alpha-ANP. DIU led to higher levels of PRA, ANG I, and ALD (p < 0.05) and lower levels of alpha-ANP (p < 0.05) compared to CON. Arginine vasopressin was not affected by the loss of PV. For the catecholamines--norepinephrine (NE) and epinephrine (EPI)--only NE was higher during exercise with DIU compared to CON (p < 0.05). For PRA and ALD, the higher levels observed during exercise with DIU could be explained both by higher resting levels and a greater increase during exercise itself. For ANG I and NE, the effect of DIU only manifested itself during exercise. In contrast, the lower alpha-ANP observed during exercise with DIU was due to the lower resting levels. These results support the hypotheses that hypohydration leads to alterations in the secretion of all of the fluid and electrolyte hormones with the exception of AVP. The specific mechanisms of these alterations remain unclear, but appear to be related directly to the decrease in PV.     

Cholinergic neuromodulation by prostaglandin D2 in canine airway smooth muscle

To determine whether prostaglandin D2 (PGD2) modulates cholinergic neurotransmission in airway smooth muscle and, if so, what the mechanism of action is, we studied bronchial segments from dogs under isometric conditions in vitro. PGD2 (10(-8)-10(-5) M) elicited dose-dependent muscle contraction, which was reduced after blockade of muscarinic receptors, so that 50% effective dose (ED50) increased from 1.3 +/- 0.3 X 10(-6) to 3.9 +/- 1.0 X 10(-6) M by atropine (10(-6) M) (mean +/- SE, P less than 0.05). Physostigmine, at a concentration insufficient to alter base-line tension (10(-8) M), enhanced the PGD2-induced contraction and decreased ED50 to 6.4 +/- 0.5 X 10(-7) M (P less than 0.05). When added at the highest doses that did not cause spontaneous contraction (1.9 +/- 0.5 X 10(-7) M), PGD2 increased the contractile response to electrical field stimulation (1-50 Hz) by 21.9 +/- 6.6% (P less than 0.001). In contrast to this effect, the response to administered acetylcholine was not affected by PGD2. On the other hand, PGD2-induced augmentation of the response to electrical field stimulation (5 Hz) was further increased from 23.6 +/- 3.0 to 70.4 +/- 8.8% in the presence of physostigmine (10(-8) M) and was abolished by atropine but not affected by the alpha-adrenergic antagonist phentolamine or the histamine H1-blocker pyrilamine. These results suggest that the contraction of airway smooth muscle induced by PGD2 is in in part mediated by a cholinergic action and that PGD2 prejunctionally augments the parasympathetic contractile response, likely involving the accelerated release of acetylcholine at the neuromuscular junction.