The effects of oestrogens and progesterone on oestrogen receptors in female rat liver.

The administration of oestradiol-17 beta or ethynyloestradiol as well as the synthetic progestogen norethisterone acetate resulted in translocation of the oestrogen receptor. Progesterone and the synthetic progestogen (+)-norgestrel were ineffective. The increases in nuclear oestrogen receptor content 1 h after injection of each steroid were similar but different subsequently. The increase with oestradiol-17 beta extended for 3--6 h and for at least 9 h with ethynyloestradiol. With norethisterone acetate, nuclear content was still increased after 24 h. Oestrogen injection resulted in cytosol receptor depletion and a 'deficit' in receptor content extending for 6 h, whereas norethisterone acetate-induced translocation was quantitative. With injections of norethisterone acetate + ethynyloestradiol the increase at 1 h and retention of the nuclear receptors were similar to that with norethisterone acetate alone. In contrast, the depletion of cytosol receptor and its restoration were similar to that seen with ethynyloestradiol alone, suggesting that norethisterone acetate did not interfere with the oestrogen receptor replenishment. Specific binding in vitro of [3H]oestradiol-17 beta in liver cytosols was inhibited by (+)-norgestrel and norethisterone acetate, but not progesterone, at concentrations of 10--100 microM. Nuclear receptors present after norethisterone acetate injection bound oestrogen with high affinity (Kd = 1.52 nM), similar to receptors of oestrogen-injected animals. In the uterus, differential retention of nuclear receptors in response to oestrogens is associated with different cellular responses. The differences in the response of the receptor system in liver to the various steroids suggests that the corresponding tissue responses may also be dissimilar. These results are discussed in relation to the problems of liver dysfunction in oral-contraceptive users.     

Neurotensin Regulation of Endogenous Acetylcholine Release from Rat Striatal Slices Is Independent of Dopaminergic Tone

The effects of neurotensin (NT) alone or in combination with the dopamine antagonist sulpiride were tested on the release of endogenous acetylcholine (ACh) from striatal slices. NT enhanced potassium (25 mM)-evoked ACh release from striatal slices in a dose-dependent manner. This effect was tetrodotoxin-insensitive, suggesting an action directly on cholinergic elements. The dopamine antagonist sulpiride (5 x 10(-5) M) significantly increased (63%) potassium-evoked ACh release from striatal slices; potassium-evoked ACh release was further increased (90%) in the presence of NT (10(-5) M) and sulpiride (5 x 10(-5) M). The second set of experiments tested the effects of 6-hydroxydopamine (6-OHDA) lesions of the substantia nigra on NT-induced increases of potassium-evoked ACh release. These lesions did not alter the NT regulation of potassium-evoked ACh release from striatal slices, but did significantly increase spontaneous (33%) and potassium-evoked (40%) ACh release from striatal slices. Striatal choline acetyltransferase activity was not affected by 6-OHDA lesions. In addition, following 6-OHDA lesions, sulpiride was ineffective in altering ACh release from striatal slices. Furthermore, evoked ACh release in the presence of the combination of NT and sulpiride was not different from that in the presence of NT alone. These results suggest that in the rat striatum, NT regulates cholinergic interneuron activity by interacting with NT receptors associated with cholinergic elements. Moreover, the NT modulation of cholinergic activity is independent of either an interaction of NT with D2 dopamine receptors or the sustained release of dopamine.     

Evidence for a dopamine receptor subtype sensitive to combination of D1 with D2 antagonist in measurement of G-protein activity using rat striatal membranes

In rat striatal membranes, various kinds of dopamine receptor agonists stimulated low-Km GTPase activity in a concentration-dependent manner. This stimulation by bromocriptine, pergolide and apomorphine was partially inhibited by sulpiride (SUL), a D2-selective antagonist, markedly inhibited by combination of SUL with SCH 23390 (SCH), a D1-selective antagonist, and not modified by SCH alone. The stimulation by BAM-1110 was resistant to SUL or SCH alone but abolished by combination of SUL with SCH. These findings suggest the presence of another subtype of a dopamine receptor in a functional in vitro bioassay system in rat striata.     

Regional adipose tissue metabolism in postmenopausal women after treatment with exogenous sex steroids

In order to evaluate the effect of exogenous sex steroids on adipose tissue metabolism, two groups of postmenopausal women were studied. In one of the groups, the effect of 50 micrograms ethinyl estradiol (EE) was investigated given orally alone and in combination with 10 mg norethisterone acetate (NET). This combination is reminiscent of an old high dose oral contraceptive. In the other group, the effect of 3 mg 17 beta-estradiol was evaluated when administered percutaneously alone and in combination with 300 mg micronized progesterone given orally. These substances and doses were chosen to provide a "physiological" hormonal influence. In the femoral region 50 micrograms EE induced an increase in LPL activity. This elevated LPL value was reversed with the addition of 10 mg NET. Moreover, during treatment with 50 micrograms EE, a decrease in norepinephrine stimulated lipolysis was seen in the abdominal region. The percutaneous administration of 17 beta-estradiol with or without micronized progesterone, however, was inert as regards subcutaneous adipose tissue metabolism. Our findings indicate, therefore, that EE in doses used in oral contraception might promote lipid accumulation in the femoral adipose tissue depot.     

Behavioral recovery after irreversible inactivation of D-1 and D-2 dopamine receptors

Irreversible inactivation of both D-1 and D-2 dopamine (DA) receptors by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) resulted in complete loss of stereotypy response to R-(-)-N-propylnorapomorphine (NPA; 0.1-1.0 mg/kg, s.c.) 24 hr later. Stereotyped sniffing recovered much more rapidly than oral behaviors. The D-2 antagonist sulpiride (200 mg/kg) and the putatively nonselective antagonist cis-flupenthixol (2 mg/kg), administered prior to EEDQ, prevented the loss of NPA-induced sniffing but only partially protected against loss of oral behaviors 24 hr later. Complete protection of both behaviors was seen after pretreatment with a combination of sulpiride and the selective D-1 antagonist SCH 23390 (1 mg/kg); pretreatment with the selective D-1 antagonist SCH 23390 alone, however, did not modify the rate of recovery of either behavioral response. The results suggest that either different populations of DA receptors mediate expression of these behaviors or stimulation of a small fraction of the total DA receptor pool may be sufficient to elicit sniffing but not oral responses. Furthermore, maintaining a normal complement of D-2 rather than D-1 receptors appears to be a critical determinant for the elicitation of these behaviors.     

Suppression of ovulation in Nile Hippopotamus (Hippopotamus amphibious) using melengestrol acetate‐treated feed or high dose Depo‐Provera injection

Analysis of fecal progestogen profiles during Depo‐Provera injection (1,200 mg; DEPO, Pfizer Inc., New York, NY), melengestrol acetate (MGA) in feed (2 or 3 mg/head/day), and a combination treatment (DEPO+MGA) are presented for nine captive female Nile hippos housed at Disney's Animal Kingdom in Florida. All tested treatments reduced fecal progestogen elevations successfully to durations consistent with prevention of ovulation for a portion of the treatment period. Percentage of treatment months with suppression of luteal phases indicative of ovulation was maximal for high‐dose MGA (91.7±13.9%) and DEPO+MGA (91.7±20.4%), followed by DEPO injection alone (69.2±13.9%) and low‐dose MGA (57.6±33.2%). Both 1,200 mg DEPO and low‐dose MGA (2.0 mg/day) treatments were insufficient to prevent an apparent seasonal breakthrough of ovarian activity from June–August 2002. Although luteal phases were observed, no females conceived during those months. Overall, in 133.5 treatment months with females housed with an adult male, one female conceived during the transition period between treatments. After cessation of contraceptive treatment, average latency to first normal ovarian cycle was 80.6±19.5 days (range = 22–179 days). Up to 12 months post‐treatment, however, successive cycles were often irregular with evidence of short periods of anovulation and shortened luteal phases in all females monitored. In conclusion, high dose and combination treatments were most successful in preventing progestogen increases indicative of ovulation in hippos. Zoo Biol 26:259–274, 2007. © 2007 Wiley‐Liss, Inc.     

Doping in sport—1. Excretion of 19-norandrosterone by healthy women, including those using contraceptives containing norethisterone

19-Norandrosterone (19-NA) is the principal urinary metabolite of the anabolic steroid nandrolone and its prohormones. The administration of these 19-nor androgens is prohibited in sport by the World Anti-Doping Agency (WADA) but, even so, adverse findings for 19-NA continue to be commonly reported. Little is known about the urinary concentrations of 19-NA that can occur in women who are not using anabolic steroids, including those using oral contraceptives containing the 19-nor progestogen norethisterone. In 2004, WADA lowered the reporting threshold for 19-NA for females from 5 to 2 ng/mL. The lack of any substantial data on 19-NA excretion in women prompted this large-scale investigation. In this investigation, single untimed urines collected from 1202 female volunteers, 38 of whom were taking norethisterone containing contraceptives, were analysed for 19-NA. None of the women was a competitive athlete and pregnancy had been excluded by a urinary test for human chorionic gonadotropin (hCG). Only one sample exceeded the 19-NA reporting threshold having a concentration of 4.1 ng/mL. This sample was from a user of a norethisterone-containing contraceptive.     

Doping in sport: 3. Metabolic conversion of oral norethisterone to urinary 19-norandrosterone

The detection of 19 norandrosterone (19-NA) in a competitor's urine sample is taken as prima facie evidence of administration of nandrolone or other 19-norsteroid but a potential problem is that administration of norethisterone, a progestogen used for menstrual disorders and for hormonal contraception, also results in the excretion of 19-NA that can exceed the laboratory reporting threshold of 2 ng/mL. The contribution of norethisterone to urinary 19-NA with and without 19-norandrostenedione, a known norethisterone tablet impurity, requires evaluation. Preparations containing, either <2 ng or 1 μg 19-norandrostenedione impurity per 5 mg of norethisterone, administered to female volunteers (n = 10) in doses comparable to those used for menstrual disorders (5 mg three times daily for 10 days), resulted in maximal 19-NA concentrations of 51 and 63 ng/mL, respectively. The maximal concentration of 19-NA, 2 h post-administration of a single 1 μg dose of 19-norandrostenedione, was 2.4 ng/mL. These results prove unequivocally that norethisterone is metabolized to 19-NA and that there is only a minor contribution from the impurity 19-norandrostenedione. Administration to women (n = 30) of a single contraceptive tablet containing norethisterone (1 mg) with one of the highest proportions of the impurity 19-norandrostenedione (∼0.5 μg, 0.05%, w/w) resulted in a urinary 19-NA concentration of 9.1 ng/mL, with a maximum concentration ratio of 19-NA to the norethisterone metabolite 3α,5β-tetrahydronorethisterone of 0.36. We provide data that should remove the need for time-consuming follow-up investigations to consider whether doping with 19-norandrogens has occurred.     

Biostimulatory effects of estrous cows and bulls on resumption of ovarian activity in postpartum anestrous Zebu (Bos indicus) cows in the humid tropics

Suckled postpartum Zebu (Bos indicus) cows were exposed either to estrous females alone or together with bulls to determine if this regimen would stimulate the resumption of ovarian activity. The study was carried out on 60 Zebu cows. The animals were allocated at parturition to 1 of 3 treatment groups. Group FB cows (n = 14) were exposed to progestogen treated females (approximately 3 cows per progestogen treated cow) and a bull for 42 d. Group F cows (n = 31) were exposed to progestogen treated females and after 21 d a bull was introduced and remained with the group until the end of the experiment 21 d later. Estrus was induced with Syncro Mate B treatment and the cows were in estrus when introduced into Groups FB and F. Group N cows (n = 15) were introduced to a bull 21 d after the treatments had begun in Groups FB and F, and the bull remained with these cows until the end of the study. On average, resumption of ovarian activity occurred in 57% (8/14), 19% (6/31) and 7% (1/15) of the cows in Group FB, F and N, respectively, within 21 d after the start of treatments. The difference was significant between Group FB and Groups F and N (P < 0.05). During the second part of the experiment, in which the bull was introduced to Groups F and N, an additional 32% (10/31) of the cows in Group F and 33% (5/15) in Group N resumed ovarian activity. No cows in Group FB resumed ovarian activity during this period. There were significant differences in body condition between cows that resumed ovarian activity and those that remained in anestrus (P < 0.05). We conclude that biostimulation effectively enhanced resumption of ovarian activity in anestrous suckled Zebu cows. This technique offers a potentially useful and practical way to improve reproductive efficiency in anestrous Zebu cattle in the tropics.     

Differences in effects of sultopride and sulpiride on dopamine turnover in rat brain

Sultopride and sulpiride are both chemically similar benzamide derivatives and selective antagonists of dopamine D2 receptors. However, these drugs differ in clinical properties. We compared the effects of sultopride and sulpiride on dopamine turnover in rats following the administration of these drugs alone or in combination with apomorphine. The administration of sultopride or sulpiride markedly accelerated dopamine turnover in the rat brain. The increase in the level of dopamine metabolites in the striatum was more marked in the sultopride-treated rats. Sulpiride affected the limbic dopamine receptors preferentially, whereas sultopride affected the striatal and the limoic dopamine receptors equally. A low dose of apomorphine induced a reduction in the concentration of dopamine metabolites in the striatum and the nucleus accumbens by approximately 55%, but not in the medial prefrontal cortex. Sultopride was more effective in preventing an apomorphine-induced reduction in dopamine metabolite levels. These results from rat experiments would model the pharmacological differences observed between sultopride and sulpiride in clinical use.     

Modulation of a neuronal network by electrical high frequency stimulation in striatal slices of the rat in vitro

The effects of the GABA(A) receptor antagonist bicuculline, the D2-like receptor antagonist sulpiride and the D1-like receptor antagonist SCH-23390 on the electrical high frequency stimulation (HFS)-evoked gamma-aminobutyric acid (GABA) and dopamine (DA) release were measured from slices of the rat striatum by means of HPLC method with electrochemical detection. HFS with 130Hz stimulated veratridine-activated GABAergic neurons resulting in an increased GABA outflow while DA outflow decreased. In the presence of the GABA(A) receptor antagonist bicuculline extracellular GABA and DA outflow were enhanced. When the competitive dopamine D2-like receptor antagonist S-(-)-sulpiride was added to incubation medium, the HFS-evoked stimulatory effect on GABA outflow declined to values found after veratridine (1microM) without HFS. After co-incubation of sulpiride and the competitive D1-like receptor antagonist R-(+)-SCH-23390, the effect of sulpiride on HFS plus veratridine-evoked GABA outflow was completely reversed. Neither sulpiride nor SCH-23390 had any influence on the effect of HFS on veratridine-induced DA outflow. No effect of HFS on glutamate outflow was observed in all experiments. These results led us to suggest that in our model HFS primarily affects GABAergic neurons. These neurons are embedded in a neuronal network with a GABA-dopamine circuit, and thus, HFS interacts with a neuronal network, not only with one neurotransmitter system or one neuron population.     

Effect of melatonin agonists and antagonists on horizontal cell spinule formation and dopamine release in a fish retina

The crucian carp retina was used to study the effects of the melatonin antagonist p697 (N-pentanoyl 2-benzyltryptamine) and the melatonin agonists [+]- and [-]-AMMTC (N-acetyl-4-aminomethyl-6-methoxy-9-methyl-1,2,3,4-tetrahydrocarbazol e) on horizontal cell spinule formation, an indicator of the state of retinal adaptation. DH97 was capable of both counteracting dark-adaptive spinule degradation and inducing light-adaptive spinule formation at the beginning of the dark phase. Addition of dopamine receptor blockers opposed the action of DH97 on spinules, with SCH 23930, a D1 dopamine receptor antagonist, being more effective than the D2 receptor antagonist sulpiride. DH97 induced a twofold increase in dopamine release. We conclude that melatonin acts as a dark signal within the teleost retina by inhibiting the dopaminergic system. In accordance with this, both enantiomers of AMMTC prevented light-induced spinule formation, and reduced dopamine release to below dark-adaptive baseline levels. We suggest that the suppression of spinule formation by AMMTC may be due to either a direct inhibitory interaction between the melatonin agonist and horizontal cell dopamine D1 receptors, or an inhibitory effect on the activity of the dopamine-releasing interplexiform cells.     

Dopaminergic metabolism in carotid bodies and high-altitude acclimatization in female rats

We tested the hypothesis that ovarian steroids stimulate breathing through a dopaminergic mechanism in the carotid bodies. In ovariectomized female rats raised at sea level, domperidone, a peripheral D2-receptor antagonist, increased ventilation in normoxia (minute ventilation = +55%) and acute hypoxia (+32%). This effect disappeared after 10 daily injections of ovarian steroids (progesterone + estradiol). At high altitude (3,600 m, Bolivian Institute for High-Altitude Biology-IBBA, La Paz, Bolivia), neutered females had higher carotid body tyrosine hydroxylase activity (the rate-limiting enzyme for catecholamine synthesis: +129%) and dopamine utilization (+150%), lower minute ventilation (-30%) and hypoxic ventilatory response (-57%), and higher hematocrit (+18%) and Hb concentration (+21%) than intact female rats. Consistent signs of arterial pulmonary hypertension (right ventricular hypertrophy) also appeared in ovariectomized females. None of these parameters was affected by gonadectomy in males. Our results show that ovarian steroids stimulate breathing by lowering a peripheral dopaminergic inhibitory drive. This process may partially explain the deacclimatization of postmenopausal women at high altitude.     

Reproductive steroid hormones and ovarian activity in felids of the Leopardus genus

Reproductive endocrine patterns were characterized in female ocelots (Leopardus pardalis; n = 3), tigrinas (Leopardus tigrinus; n = 2), and margays (Leopardus wiedii; n = 2) housed in captivity in southern Brazil. Females were maintained as singletons and exposed to natural fluctuations in photoperiod. Cyclic changes in ovarian steroids were monitored by analyzing estrogen and progestogen metabolites in fecal samples collected five times weekly for 14 to 18 months. Based on intervals between fecal estrogen peaks, mean (± SEM) duration of the estrous cycle was 18.4 ± 1.6 days for the ocelots (range, 7–31 days; n = 75 cycles), 16.7 ± 1.3 days for the tigrinas (range, 11–27 days; n = 23 cycles), and 17.6 ± 1.5 days for the margays (range, 11–25 days; n = 32 cycles). Fecal progestogen analyses combined with two laparoscopic observations of the ovaries confirmed that ocelots and tigrinas did not ovulate spontaneously. In contrast, non‐mating–induced luteal phases of 40.1 ± 6.3 days in duration (range, 30–60 days) were observed frequently in both margays. There was no evidence of gonadal seasonality in margays in either follicular or luteal activity. In ocelots, cyclic changes in estrogen excretion were observed during each month of the year; however, only one female cycled continuously. In the other two ocelots, periods of acyclicity of several months’ duration were observed. It was not possible to conclude whether tigrinas were aseasonal because estrous cyclicity was observed in only one of two individuals. In the female that cycled, a 3‐month period of acyclicity was observed in the late fall/early winter. These data demonstrate similarities among three felid species of the genus Leopardus, including evidence they are polyestrous but experience unexplained periods of ovarian inactivity. Only the margays differed by exhibiting occasional spontaneous, non‐mating–induced ovulations. Historically, these species have not bred well in captivity. However, it is hoped that understanding the biological similarities and differences among them could lead to improved management strategies that may one day result in increased reproductive success. Zoo Biol 20:103–116, 2001. © 2001 Wiley‐Liss, Inc.     

In vivo CREB phosphorylation mediated by dopamine and NMDA receptor activation in mouse hippocampus and caudate nucleus

The pattern of CREB phosphorylation was investigated in the caudate nucleus and hippocampus 10 min or 3 h after i.p. injection of dopamine or NMDA receptor agonists alone, or in combination with antagonists. Ten minutes after C57BL/6 J mice were injected with either the dopamine D1 receptor agonist SKF-38393 hydrobromide or NMDA, immunoreactivity of phosphorylated CREB (pCREB) was significantly increased in all parts of the caudate nucleus but not in hippocampal regions. However, 3 h after the injection of SKF-38393, pCREB levels in the caudate nucleus did not differ significantly from the pCREB levels in control animals, whereas pCREB levels were still elevated 3 h after NMDA injection. Except for the D1 receptor antagonist SCH-23390, which induced CREB phosphorylation in the caudate nucleus, dopamine and NMDA receptor antagonists had little effect on pCREB levels by themselves. However, the NMDA receptor antagonist CGS-19755 injected i.p. blocked both the NMDA- and SKF-38393-induced rise of pCREB levels in the caudate nucleus. Similarly, the D1 receptor antagonist SCH-23390 inhibited the effects produced by SKF-38393 or NMDA. Interestingly, the D2 receptor antagonist sulpiride also blocked the SKF-38393-triggered rise of pCREB. The results demonstrated that NMDA and dopamine receptors modulate pCREB levels in the caudate nucleus and suggest mutual permissive roles for both receptors.     

Progesterone and norethisterone have different effects on tachykinin-like immunoreactivity in rat cortex and striatum

OBJECTIVE: The purpose of this study was to investigate the effects of progesterone and the most commonly prescribed synthetic progestogen, norethisterone, on regional immune-like reactivity of neuropeptide Y (NPY), substance P (SP), neurokinin A (NKA) and neurotensin (NT) in brains of female ovariectomized estradiol-substituted rats. Results: Norethisterone+estradiol-treated rats had 44% lower SP levels compared with estradiol-only-treated in frontal cortex and 20% lower NKA levels in comparison with progesterone+estradiol-treated in frontal cortex. Progesterone+estradiol-treated rats had 66% lower SP levels in striatum in comparison with both estradiol-only-treated and norethisterone+estradiol-treated. No significant results were found for NPY and NT. CONCLUSION: Progesterone and the synthetic progestogen, norethisterone, have different effects on SP- and NKA-like immunoreactivity in rat cortex and striatum.The effects of NET on SP- and NKA-like immunoreactivity in frontal cortex may contribute to the mood effects ascribed to this progestogen in clinical usage.     

Genotoxicity evaluation of norethisterone acetate

Genotoxic evaluation of a commonly used progestogen, norethisterone acetate, was undertaken using a combination of short-term in vitro and in vivo assays. The clastogenic potentiality of norethisterone acetate was evident from the chromosome aberrations and sister chromatid exchanges induced both with and without S9 mix in cultured human lymphocytes and also from the increased frequency of micronuclei formation and sister chromatid exchanges in mice. However, in the Ames Salmonella assay, both with and without S9 mix and in host-mediated assay, norethisterone acetate was unable to cause any significant increase/decrease in the His⁺ revertants/plate.     

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 less than 0.001; n = 6). This increase was antagonized by treatment with sulpiride (15 mg kg-l). Incubation of the arterial tissue with bromocriptine (50 micrograms ml-1) in vitro also stimulated PGI2 release. Mepacrine (160 micrograms ml-1) significantly decreased both basal and stimulated PGI2 release. Incubation of myometrial tissue from pregnant rats with bromocriptine (50 micrograms ml-1) in vitro significantly decreased PGI2 release from 1.25 +/- 0.07 to 0.60 +/- 0.08 ng/mg wet tissue (P less than 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 effect 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.     

Effect of Guanine Nucleotides on Dopaminergic Agonist and Antagonist Affinity for [3H]Sulpiride Binding Sites in Rat Striatal Membrane Preparations

Abstract: [³H]Sulpiride bound to rat striatal membrane preparations with a saturable, high affinity component. This binding was displaced potently by dopamine antagonists (both classic neuroleptics and the benzamide, sulpiride) and less potently by dopamine agonists. GTP and its stable analogue Gpp(NH)p did not affect [³H]sulpiride binding to the membranes but altered the affinity for dopaminergic agonists. This effect was specific in that antagonist binding was not affected and only GTP, GDP, and Gpp(NH)p produced the effect. Similar alterations in ligand binding affinity caused by guanine nucleotides have been observed for binding sites linked to an adenylate cyclase. Such an interpretation for the case of [³H]sulpiride is contrary to suggestions that sulpiride labels only those dopamine receptors that are not cyclase linked.     

Simultaneous measurement of glutamate and dopamine release from isolated guinea pig cochlea

Glutamate is proved to be a neurotransmitter in the mammalian cochlea, transmitting signals between the inner hair cells and the afferent cochlear nerve terminals. The transmission in this synapse is modulated by the lateral olivocochlear efferent fibers by releasing dopamine and other neurotransmitters. This study undertakes to measure simultaneously the release of dopamine and glutamate from isolated guinea pig cochleae. We combined the in vitro microvolume superfusion method, that uses liquid scintillation analysis, to measure [3H]dopamine with high pressure liquid chromatography (HPLC) to determine the glutamate content of the superfusate at rest and during stimulation. The release of both neurotransmitters was significantly increased when electrical field stimulation was applied at a 10 Hz rate. The nonselective sodium-channel inhibitor tetrodotoxin (TTX) at 1 microM completely blocked the effect of stimulation, indicating the neural origin of both dopamine and glutamate. The dopamine receptor antagonist sulpiride at 100 microM and the dopamine receptor agonist bromocriptine at 20 microM did not change the release of glutamate. In contrast, both bromocriptine and sulpiride significantly increased the stimulation-evoked release of dopamine. The effect of sulpiride is most likely due to the blockade of dopamine autoreceptor. Possible explanations why bromocriptine increased the release include: (1) its partional agonist activity; (2) desensitizations of dopamine autoreceptors; or (3) the higher D1 receptor activity of bromocriptine than sulpiride. This study could provide further insights about the role of dopamine and glutamate in cochlear neurotransmission.