Role of cyclic AMP in the action of dopaminergic D2 receptors of some endocrine glands in rats

Short-term and long-term effects of bromocriptine mesylate (10 mg/kg i.p.) on cyclic AMP contents of the liver and some endocrine glands have been investigated in the presence and absence of sulpiride (10 mg/kg i.p.). Results revealed that bromocriptine caused significant elevations in the cyclic AMP contents of the liver and reduction in its adrenocortical content. Bromocriptine effect on the adrenal cortex was antagonized by sulpiride, whereas its effect on the liver was not changed. Bromocriptine did not change the, cyclic AMP content in the thyroid gland or the ovary.     

Role of Na+/K+-stimulated adenosine triphosphatase in the action of dopaminergic-D2 receptors of the liver in rats

The dopamine receptor agonist, bromocriptine, in a dose of 10 mg/kg i.p. for 14 days, in rats caused a significant increase in liver Na⁺/K⁺-ATPase activity, whereas sulpiride, a dopamine receptor antagonist, in a dose of 10 mg/kg, i.p. for 14 days, in rats, caused a significant decrease in liver Na⁺/K⁺-ATPase activity. Injection of bromocriptine and sulpiride simultaneously in a group of rats, under the same conditions and using the same doses caused a complete block of both stimulatory activity of bromocriptine and inhibitory activity of sulpiride on liver Na⁺/K⁺-ATPase activity. It is suggested that Na⁺/K⁺-ATPase may have a role in the action of dopaminergic-D₂ receptors.     

Effects of bromocriptine on cyclic-GMP contents of some endocrine glands and liver in rats

Cyclic GMP contents of the thyroid gland and ovary were significantly increased in response to single and multiple treatments of bromocriptine, an effect which was antagonized by sulpiride. Liver and adrenocortical cyclic GMP levels have not been changed by bromocriptine although sulpiride alone induced a significant reduction. The data may indicate the presence of D₂ receptors in ovary and thyroid gland that are related to cyclic GMP.     

Effect of bromocriptine on prostacyclin release and cyclic nucleotides on rat aortic and uterine tissues

The effect of bromocriptine mesylate on cyclic nucleotides and PGI₂ release by rat aortic and uterine tissues was investigated. Treatment of rats with bromocriptine (10 mg kg⁻¹ I.P. daily for 14 days) increased PGI₂ release by the thoracic aorta from 0.67 ± 0.02 to 1.4 ± 0.03 ng/mg wet tissue (P < 0.001; n = 6). This increase was antagonized by treatment with sulpiride (15 mg kg⁻¹). Incubation of the arterial tissue with bromocriptive (50 ug ml⁻) in vitro also stimulated PGI₂ release. Mepacrine (160 μg ml⁻) significantly decreased both basal and stimulated PGI₂ release. Incubation of myometrial tissue from pregnant rats with bromocriptine (50 μg ml⁻¹) in vitro significantly decreased PGI₂ release from 1.25 ± 0.07 to 0.60 ± 0.08 ng/mg wet tissue (P < 0.05, n = 6).It also elevated uterine cAMP from 40 ± 2 to 64 ± 3 pmoles/100 mg wet tissue. Both effects were antagonized by sulpiride. Bromocriptine did not affect uterine cGMP or the cyclic nucleotides in the aorta. It is concluded that the increase in aortic PGI₂ was mediated via activation of dopamine D-2 receptors that stimulate phospholipase A₂ enzyme. The decrease in myometrial PGI₂ release may be related to the increase in uterine cAMP resulting from activation of dopamine D-1 receptors. Previous studies suggested a role for PGI₂ in implantation in the rat. The results suggest that the inhibitory effèct on uterine PGI₂ may underlie the reported inhibition of bromocriptine on implantation. On broad basis, the decrease in uterine PGI₂ together with the reported luteolytic effect of bromocriptine point to a potential role for the compound in postcoital contraception.     

Prolactin binding in rat mammary gland during pregnancy and lactation

Lactogenic hormones from the placenta and pituitary are primarily responsible for the growth and function of the mammary gland during pregnancy and lactation. In the present study we describe the optimal conditions for the measurement of ¹²⁵I-labeled ovine prolactin binding to mammary gland slices of pregnant and lactating rats. Prolactin binding is saturable (K_{d approx. 2.36 · 10^?9 M)}, hormone specific and destroyed by proteases. The hormonal environments of pregnancy and lacation dramatically influence the availability and measurement of prolactin binding sites. Whereas binding consistently appears to be low in mammary glands removed from rats during pregnancy, binding levels rise 7–8-fold shortly after birth and remain high during the 22 days of lactation. However, the removal of the ovaries and gravid uteri at specific times during pregnancy results in prompt 3–6-fold increase in prolactin binding. Elevated levels in potential prolactin binding capacity appear in mammary tissue coincident with the reported rise in serum rat placental lactogen between the eight and eleventh days. We suggest that high levels of this lactogenic hormone promote the appearance of prolactin binding sites during pregnancy and mask the sites such that they are not available for measurement in vitro.     

Prolactin cycles in sheep under constant photoperiod: evidence that photorefractoriness develops within the pituitary gland independently of the prolactin output signal

The present study investigated photorefractoriness in the prolactin (PRL) axis in hypothalamopituitary-disconnected (HPD) sheep exposed to prolonged long days. In experiment 1, HPD Soay rams transferred from short (8L:16D) to long (16L:8D) days for 48 wk to induce a cycle of activation, decline (photorefractoriness), and reactivation in PRL secretion were treated chronically with bromocriptine (dopamine-receptor agonist) or vehicle from the onset of photorefractoriness. Bromocriptine (0.01-0.04 mg kg-1 day-1; 12-24 wk of long days) blocked PRL release and caused a rebound response after the treatment, but it had no effect on the long-term PRL cycle (posttreatment PRL minimum, mean +/- SEM, 35.3 +/- 0.6 and 37.0 +/- 0.4 wk for bromocriptine and control groups, respectively; not significant). In experiment 2, HPD rams were treated with sulpiride (dopamine-receptor antagonist) during photorefractoriness. Sulpiride (0.6 mg/kg twice daily; 22-30 wk of long days) induced a marginal increase in blood PRL concentrations, but again, it had no effect on the long-term PRL cycle (PRL minimum, 37.9 +/- 0.4 and 37.6 +/- 0.9 wk for sulpiride and control groups, respectively; not significant). The 24-h blood melatonin profile consistently reflected the long-day photoperiod throughout, and blood FSH concentrations were minimal, confirming the effectiveness of the HPD surgery. The results support the conclusion that photorefractoriness is regulated at the level of the pituitary gland independently of the PRL output signal.     

Binding of high-density lipoproteins to luteal membranes: the role of prolactin, luteinizing hormone, and circulating lipoproteins

Ovarian and adrenal membranes from immature gonadotropin-primed rats, treated with 4-amino-pyrazolopyrimidine (4APP) to reduce endogenous lipoprotein levels, displayed higher binding of porcine high-density lipoprotein (HDL) when compared to control rats. Immature, hypophysectomized (HYPOX) rats bearing corpora lutea (CL) on Day 5 after ovulation had lower levels of serum progesterone and reduced capacity for HDL and human chorionic gonadotropin (hCG) binding to ovarian membranes when compared with intact animals. Hypophysectomy also reduced the number of HDL binding sites in adrenal membranes. Treatment of HYPOX animals with luteinizing hormone (LH) and prolactin (Prl) alone or in combination increased the HDL binding sites in the ovary relative to HYPOX-untreated rats. Neither hormone affected binding to adrenals, where only adrenocorticotropic hormone (ACTH) enhanced HDL binding. LH treatment reduced the serum progesterone levels and hCG binding to the ovaries, whereas Prl administration increased progesterone levels with no effect on hCG binding. We conclude from this study that HDL binding in the luteinized ovary is regulated by Prl and LH and circulating lipoproteins, whereas in adrenals it is regulated by ACTH and circulating levels of lipoproteins.     

Peripheral benzodiazepine receptors in isolated human pancreatic islets

Peripheral benzodiazepine receptors have been shown in some endocrine tissues, namely the testis, the adrenal gland, and the pituitary gland. In this work we evaluated whether peripheral benzodiazepine receptors can be found in the purified human pancreatic islets and whether they may have a role in insulin release. Binding of the isoquinoline compound [³H]1-(2-chlorophenyl-N-methyl-1-methyl-propyl)-3-isoquinolinecarboxamide ([³H]PK-11195), a specific ligand of peripheral benzodiazepine receptors, to cellular membranes was saturable, and Scatchard's analysis of the saturation curve demonstrated the presence of a single population of binding sites, with an affinity constant value of 9.20 ± 0.80 nM and a maximum number of binding sites value of 8913 ± 750 fmol/mg of proteins. PK-11195 and 7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1,4-benzodiazepin-2-on (Ro 5-4864) significantly potentiated insulin secretion from freshly isolated human islets at 3.3 mM glucose. These results show the presence of peripheral benzodiazepine receptors in purified human pancreatic islets and suggest their role in the mechanisms of insulin release. J. Cell. Biochem. 64:273–277. © 1997 Wiley-Liss, Inc.     

Prolactin and Growth Hormone Binding in Mammary and Liver Tissue of Lactating Cows

Abstract

A radioligand/receptor binding assay was developed using homologous hormones to distinguish between bovine growth hormone (bGH) and bovine prolactin (bPRL) receptors in liver and mammary tissue of lactating cows. Mammary and liver tissues were homogenized in 0.3 M sucrose and centrifuged at 100,000 x g over a 1.3 M sucrose density gradient. Membranes from the 0.3 - 1.3 M sucrose interface were incubated with 1 ng of iodinated bGH or bPRL for 20 h at 22°C in the presence of increasing concentrations of native bGH or bPRL. High affinity receptor binding sites were found for bPRL in liver and mammary tissue membranes (Ka=3.2 and 1.3 × 10⁸ 1/mol with 34 and 63 fmol receptors/mg liver and mammary membrane protein, respectively) and for bGH only in liver tissue (Ka=1.8 × 10⁹ 1/mol, 18 fmol receptors/mg membrane protein). Receptor number estimates were 3 and 11 times higher in mammary and liver tissue using a heterologous hGH system indicating that heterologous systems may overestimate the number of receptors in bovine tissue. The absence of demonstratable bGH receptors in lactating bovine mammary tissue supports in vitro results of others with isolated mammary tissue indicating that the positive effect of bGH on milk production in intact cows is via an indirect mechanism.     

Evidence for dopaminergic regulation of prolactin and a luteotropic complex in the ferret

The role of dopaminergic agents in prolactin (Prl) release and the luteotrophic role of Prl and luteinizing hormone (LH) were investigated in pseudopregnant female ferrets. A single injection of the dopamine antagonist pimozide (0.63 mg/kg) resulted in a tenfold elevation of plasma Prl in anestrous females. Subcutaneous injection of pimozide on alternate days from Day 2 through Day 16 of pseudopregnancy elevated both Prl and progesterone levels. Daily treatment with the dopamine agonist 2 alpha-bromoergocryptine (bromocriptine, 4 mg/kg), from Day 2 through Day 16 of pseudopregnancy lowered levels of both plasma Prl and progesterone. Neither pimozide nor bromocriptine had a direct effect on progesterone secretion by luteal cells in vitro. Daily intraperitoneal administration of a monoclonal antibody against gonadotropin-releasing hormone from Day 2 through Day 10 of pseudopregnancy lowered both plasma LH and progesterone, but had no effect on plasma Prl concentrations. Daily administration of equine antisera against bovine LH or 100 IU of human chorionic gonadotrophin to pseudopregnant ferrets lowered progesterone levels. It is concluded that Prl release is influenced by dopaminergic compounds, and both Prl and LH are required for luteal maintenance in the ferret.     

Prolactin stimulation of prolactin receptors in rat liver.

Hypophysectomy of the female rat results in a loss of prolactin receptors from the liver. Seventy percent of specific prolactin binding is lost within 24 hours; by 48 hours receptor levels are less than 5% of those found in livers from intact rats. A single dose of ovine prolactin to hypophysectomized rats causes a partial restoration of prolactin receptors between 12 and 18 hours after injection. As little as 0.5 mg prolactin significantly increases receptors, while doses above the 2 mg optimum are apparently less effective. These restored receptor sites are unaltered in their affinity for prolactin. Estradiol (E₂), progesterone, hydrocortisone, triiodothyronine (T₃) or E₂ plus T₃ could not mimic the prolactin effect. Neither combinations of prolactin with E₂ or T₃ nor repeated daily injections of prolactin alone increase receptors more than does a single prolactin injection. It appears that prolactin modulates the level of its own receptor in rat liver.     

Histomorphic changes following chronic adrenocortical activation and inhibition in the pigeon

Gravimetric and histologic modifications in the pigeon were studied following chronic therapy with ACTH and metopirone (SU 4885) for a period of 15 days. The organs studied were proventriculus, duodenum, heart, kidney, salivary gland, pancreas, liver, uropygial gland, thymus, spleen, bursa fabricii, testis, ovary, islets of Langerhans, adenohypophysis, thyroid and interrenal and chromaffin tissue of the adrenal gland. Induced states of hyper- and hypoadrenocorticalism elicited pathomorphic changes in endocrine and reproductive systems and some other organs of the pigeon. There were many differences and similarities in the nature of response of some organs following the two experimental conditions. Many of these cellular interactions might have resulted from alteration of interrenal function in the pigeon.     

Proiactin Binding in Rat Langerhans Islets

Abstract

Membrane preparations of collagenase-dispersed Langerhans islets of female Wistar rats exhibit specific binding sites for ¹²⁵I labelled ovine prolactin (¹²⁵I-oPrl). Almost negligible binding was detected in islets of male animals. The binding is a saturable and time-temperature dependent process, equilibrium being reached after 16 h incubation at 0°C. The bound oPrl is not displaceable by hFSH, hLH, bGH or hGH. In contrast with other cell fractions, the 12,000 g pellet accounts for more than 80% of the specific binding of ¹²⁵I-oPrl. Scatchard plots of data obtained in saturation studies indicate a single class of binding sites with K_a = 0.21 × 10¹⁰ M^-1. Protein and phospholipid moieties are essential for the receptor activity, since after trypsin or phospholipase C digestions marked loss of binding was verified. In islets of streptozotocin diabetic rats a marked reduction in the number of binding sites was observed. These findings may suggest that some of the actions of prolactin on endocrine pancreas could be explained by its specific interaction with islet cell membranes.     

Neuroleptics Up-Regulate Adenosine A2a Receptors in Rat Striatum: Implications for the Mechanism and the Treatment of Tardive Dyskinesia

Abstract: Neuroleptics, which are potent dopamine receptor antagonists, are used to treat psychosis. In the striatum, dopamine subtype-2 (D₂) receptors interact with high-affinity adenosine subtype-2 (A_2a) receptors. To examine the effect of various neuroleptics on the major subtypes of striatal dopamine and adenosine receptors, rats received 28 daily intraperitoneal injections of these drugs. Haloperidol (1.5 mg/kg/day) increased the density of striatal D₂ receptors by 24% without changing their affinity for [³H]sulpiride. Haloperidol increased the density of striatal A_2a receptors by 33% (control, 522.4 ± 20.7 fmol/mg of protein; haloperidol, 694.6 ± 23.6 fmol/mg of protein; p < 0.001) without changing their affinity for [³H]CGS-21680 (control, 19.2 ± 2.2 nM; haloperidol, 21.4 ± 2.3 nM). In contrast, haloperidol had no such effect on striatal dopamine subtype-1 (D₁) and adenosine subtype-1 (A₁) receptors. Binding characteristics and the pharmacological displacement profile of the increased [³H]CGS-21680 binding sites confirmed them as A_2a receptors. Comparing different classes of neuroleptics showed that the typical neuroleptics haloperidol and fluphenazine (1.5 mg/kg/day) increased D₂ receptor densities, whereas the atypical neuroleptics sulpiride (100 mg/kg/day) and clozapine (20 mg/kg/day) did not (control, 290.3 ± 8.7 fmol/mg of protein; haloperidol, 358.1 ± 6.9 fmol/mg of protein; fluphenazine, 381.3 ± 13.6 fmol/mg of protein; sulpiride, 319.8 ± 18.9 fmol/mg of protein; clozapine, 309.2 ± 13.7 fmol/mg of protein). Similarly, the typical neuroleptics increased A_2a receptor densities, whereas the atypical neuroleptics did not (control, 536.9 ± 8.7 fmol/mg of protein; haloperidol, 687.9 ± 28.0 fmol/mg of protein; fluphenazine, 701.1 ± 31.6 fmol/mg of protein; sulpiride, 563.3 ± 27.2 fmol/mg of protein; clozapine, 550.9 ± 40.9 fmol/mg of protein). There were no differences in affinities for [³H]CGS-21680 or [³H]sulpiride among the various treatment groups. This study demonstrates that typical neuroleptics induce comparable up-regulation in both striatal D₂ and A_2a receptors. Thus, A_2a receptors might be a pharmacologic target for the development of novel therapeutic strategies to minimize the adverse effects of antipsychotic treatment.     

Effect of temperature and ionic environment on the specific binding of 3H(—)sulpiride to membranes from different rat brain regions

Sulpiride is an antipsychotic drug endowed with the properties of a dopamine antagonist. The failure of sulpiride to inhibit neostriatal dopamine stimulated adenylate cyclase activity indicated that this drug is a selective D₂ receptor antagonist. In this study we used a novel synthesized ²H(—)sulpiride with very high specific activity (72 Ci/mol) and characterized the temperature sensitivity of the binding sites labeled by this compound. Kinetic analysis of ³H(—)sulpiride binding in rat striatum showed unstable behavior when incubation was performed at 37 or 30°C. However when experiments were carried out at 15 or 10°C, binding reached a stable steady-state within 10 min. Scatchard analysis of binding isotherms obtained at 10°C showed a 5-fold increase in the maximum number of binding sites and a decrease in K_d values to one-third those obtained at 37°C. Pharmacological characterization of the binding sites labeled by ³H(—)sulpiride at 10°C showed a greater affinity for antagonists but not for agonists than 37°C. Under both experimental condition, ³H(—)sulpiride binding sites were Na⁺ and GTP-sensitive. The temperature sensitive binding phenomenon appeared to be area specific. ³H(—)sulpiride binding sites in tissues other than from striatum were influenced less or not at all by changes in incubation temperature.     

Pathological effects of the radiation protector WR-151327 in mice

The systemic effects of the radiation protective agent, S-3-(3-methylaminopropylamino) propylphosphorothioic acid (WR-151327), were studied in unirradiated B6CF1 male mice. Fifty mice were injected intraperitoneally with 540 mg/kg WR-151327, and groups of five mice were sacrificed at 14-day intervals up to and including 140 days post-treatment. Ten mice served as sham-injected controls. A necropsy was performed and gross morphological abnormalities were noted. Tissues (brain, eyes, harderian gland, salivary glands, sternal bone marrow, thyroid, lung, thymus, esophagus, trachea, skeletal muscle, heart, liver, kidney, adrenal gland, spleen, small intestine, pancreas, and testes) were fixed in 10% formalin, embedded in paraffin, and sectioned. Slides were routinely stained with hematoxylin and eosin while Alizarin red stain was used to test specifically for the presence of calcium salts. Histopathological effects of WR-151327 were restricted to the testes, salivary gland, and pancreas. The caudal pole of the testes was observed to undergo progressive changes from coagulation necrosis to dystrophic calcification. The cells of the submandibular salivary gland showed mainly hyperchromatic nuclei while the pancreas showed enlarged islets of Langerhans.     

Dopamine receptors in the rat brain: quantitative autoradiographic localization using [3H]sulpiride

In vitro labeling of tissue sections with [³H]sulpiride has been utilized in the present study to autoradiographically localize D₂-dopamine receptors in the rat brain. Preliminary biochemical studies, using slide-mounted tissue sections, were performed to define the optimal labeling conditions for this binding. Autoradiograms were generated by apposition of the labeled tissue sections to tritium-sensitive film. Specific binding sites for [³H]sulpiride were localized to the caudate-putamen, nucleus accumbens, olfactory tubercle, glomerular layer of the olfactory bulb, pituitary, laminae I and III of the entorhinal cortex, substantia nigra, lateral mammillary nucleus and the stratum-lacunosum moleculare of the hippocampus. The high selectivity of [³H]sulpiride for the D₂-dopamine receptor indicates that it is a valuable tool for the autoradiographic localization and quantitation of neuroleptic receptors.     

A Comparative Study of Lactogenic Hormone Binding Sites in the Adrenal Gland,Ovary and Kidney of the Lactating Cow

Abstract

The specific binding of ¹²⁵I-oPRL to microsomal fractions from the adrenal gland, ovary and kidney of the lactating cow was significantly lower than binding of iodinated hGH. In addition, the ability of oPRL to compete with iodinated hGH as compared to hGH, was 50-fold lower in the adrenal gland 35-fold lower in the ovary and 18-fold lower in the kidney. These results are similar to those obtained in the mammary gland and liver, whereas the ability of oPRL to compete with iodinated hGH was 90-fold lower, as compared to hGH. In the kidney the difference between the binding of iodinated hGH and iodinated oPRL was smaller. Results obtained with a solubilized kidney microsomal fraction also show a slightly higher affinity for oPRL than in other tissues. Thus the phenomena of differential affinities of oPRL and hGH to lactogenic hormone binding sites, characterizes most lactogenic hormone target tissues in the lactating cow. The distribution of these sites in different parts of the tissues was also studied. In the adrenal gland, the binding capacity in the cortex was 8-fold higher than in the medulla. In the ovary most of the binding sites were found in the corpus luteum, while in the kidney the binding capacity was higher in the cortex as compared to the medulla.     

Effect of bromocriptine on lipid plasma levels in rats

Results show that bromocriptine induced marked alterations in plasma levels of cholesterol and lipids in response to acute and chronic administrations in rats. Two hours after an I.P. dose of 10 mg/kg, bromocriptine mesylate caused significant reductions in plasma levels of total high density lipoprotein (HDL) and high density lipoprotein cholesterol (HDL cholesterol). At a dose of 20 mg/kg, bromocriptine mesylate induced significant elevations in plasma levels of total cholesterol, total HDL, HDL cholesterol, total low density lipoproteins (LDL), and low density lipoprotein cholesterol (LDL cholesterol). Injected at a dose of 4 or 10 mg/kg daily for 14 consecutive days, bromocriptine mesylate caused significant increases in plasma levels of total cholesterol, LDL cholesterol and total LDL whereas the levels of HDL cholesterol, total HDL triglycerides (TG) were reduced. At a dose of 20 mg/kg all parameters were significantly increased. Marked hyperglycaemia was noticed in response to doses of 10, 15 and 20 mg/kg injected daily for 14 consecutive days or 2 hrs after a single administration of 15 mg/kg. Plasma insulin activity was reduced 2 hours after injection of bromocriptine at a dose of 15 mg/kg Likewise, a significant reduction in plasma insulin activity was observed in response to daily I.P. injections of bromocriptine at a dose of 15 mg/kg. Hyperglycaemic and hypoinsulinaemic effects of bromocriptine (acute and chronic) were markedly decreased when sulpiride, a dopaminergic D2 antagonist, was injected at an I.P. dose of 10 mg/kg before bromocriptine. Plasma ACTH activity was significantly increased in response to bromocriptine (15 mg/kg I.P.) in acute and chronic experiments. This effect was markedly diminished when sulpiride was injected prior to bromocriptine. In conclusion, bromocriptine induced marked elevations in plasma levels of total cholesterol and lipids which are likely to be related to hyperglycaemic and hypoinsulinaemic effects.     

Human growth hormone binds to lactogenic receptors in bovine, ovine and rat adrenals

The distribution of 125I radioactivity in the liver, kidneys, adrenals and serum of male rats was measured 10 minutes after an intravenous bolus of 125I-labelled human growth hormone (hGH) was administered in the presence or absence of a large excess of ovine growth hormone or ovine prolactin. The hGH binding sites in the adrenals had displacement properties characteristic of lactogenic receptors, whereas those in the liver had displacement properties characteristic of somatogenic receptors. Bovine and ovine adrenal microsomal membrane fractions contained high affinity (Ka = 1.4-3.3 nM-1) binding sites for hGH which showed ligand specificity typical of lactogenic receptors. It is concluded that the hGH binding site in the adrenal gland is a classical lactogenic receptor and that this tissue is a convenient and rich (42.6 +/- 6.4 fmol hGH specifically bound/mg protein) source of receptor suitable for further characterization.