A genetic determinant of the striatal dopamine response to alcohol in men.

Excessive alcohol use, a major cause of morbidity and mortality, is less well understood than other addictive disorders. Dopamine release in ventral striatum is a common element of drug reward, but alcohol has an unusually complex pharmacology, and humans vary greatly in their alcohol responses. This variation is related to genetic susceptibility for alcoholism, which contributes more than half of alcoholism risk. Here, we report that a functional OPRM1 A118G polymorphism is a major determinant of striatal dopamine responses to alcohol. Social drinkers recruited based on OPRM1 genotype were challenged in separate sessions with alcohol and placebo under pharmacokinetically controlled conditions, and examined for striatal dopamine release using positron emission tomography and [(11)C]-raclopride displacement. A striatal dopamine response to alcohol was restricted to carriers of the minor 118G allele. To directly establish the causal role of OPRM1 A118G variation, we generated two humanized mouse lines, carrying the respective human sequence variant. Brain microdialysis showed a fourfold greater peak dopamine response to an alcohol challenge in h/mOPRM1-118GG than in h/mOPRM1-118AA mice. OPRM1 A118G variation is a genetic determinant of dopamine responses to alcohol, a mechanism by which it likely modulates alcohol reward.

Functional expression and FRET analysis of green fluorescent proteins fused to G-protein subunits in rat sympathetic neurons.

1. cDNA constructs coding for a yellow-emitting green fluorescent protein (GFP) mutant fused to the N-terminus of the G-protein subunit beta 1 (YFP-beta 1) and a cyan-emitting GFP mutant fused to the N-terminus of the G-protein subunit gamma 2 (CFP-gamma 2) were heterologously expressed in rat superior cervical ganglion (SCG) neurons following intranuclear injection of the tagged subunits. The ability of the tagged subunits to modulate effectors, form a heterotrimer and couple to receptors was characterized using the whole-cell patch-clamp technique. Fluorescent resonance energy transfer (FRET) was also measured to determine the protein-protein interaction between the two fusion proteins. 2. Similar to co-expression of untagged beta 1/gamma 2, co-expression of YFP-beta 1/gamma 2, beta 1/CFP-gamma 2, or YFP-beta 1/CFP-gamma 2 resulted in a significant increase in basal N-type Ca(2+) channel facilitation when compared to uninjected neurons. Furthermore, the noradrenaline (NA)-mediated inhibition of Ca(2+) channels was significantly attenuated. 3. Co-expression of YFP-beta 1/CFP-gamma 2 with G-protein-gated inwardly rectifying K(+) channels (GIRK1 and GIRK4) resulted in tonic GIRK currents that were blocked by Ba(2+). 4. The ability of the tagged subunits to form heterotrimers was tested by co-injecting either tagged or untagged G beta 1 and G gamma 2 with excess G alpha(oA) cDNA. Under these conditions, the NA-mediated Ca(2+) current inhibition was significantly decreased when compared to uninjected neurons. 5. Coupling to the alpha 2-adrenergic receptor was reconstituted in neurons expressing pertussis toxin (PTX)-insensitive G alpha(oA) and either tagged or untagged G beta 1 gamma 2 subunits. Application of NA to PTX-treated cells resulted in a voltage-dependent inhibition of N-type Ca(2+) currents. 6. FRET measurements in the SCG revealed an in vivo interaction between YFP-beta 1 and CFP-gamma 2. Co-expression of untagged beta 1 significantly decreased the interaction between the two fusion proteins. 7. In summary, the attachment of GFP mutants to the N-terminus of G beta 1 or G gamma 2 does not qualitatively impair their ability to form a heterotrimer, modulate effectors (N-type Ca(2+) and GIRK channels), or couple to receptors.

A voltage-independent calcium current inhibitory pathway activated by muscarinic agonists in rat sympathetic neurons requires both Galpha q/11 and Gbeta gamma.

Calcium current modulation by the muscarinic cholinergic agonist oxotremorine methiodide (oxo-M) was examined in sympathetic neurons from the superior cervical ganglion of the rat. Oxo-M strongly inhibited calcium currents via voltage-dependent (VD) and voltage-independent (VI) pathways. These pathways could be separated with the use of the specific M(1) acetylcholine receptor antagonist M(1)-toxin and with pertussis toxin (PTX) treatment. Expression by nuclear cDNA injection of the regulator of G-protein signaling (RGS2) or a phospholipase Cbeta1 C-terminal construct (PLCbeta-ct) selectively reduced VI oxo-M modulation in PTX-treated and untreated cells. Expression of the Gbetagamma buffers transducin (Galpha(tr)) and a G-protein-coupled-receptor kinase (GRK3) construct (MAS-GRK3) eliminated oxo-M modulation. Activation of the heterologously expressed neurokinin type 1 receptor, a Galpha(q/11)-coupled receptor, resulted in VI calcium current modulation. This modulation was eliminated with coexpression of Galpha(tr) or MAS-GRK3. Cells expressing Gbeta(1)gamma(2) were tonically inhibited via the VD pathway. Application of oxo-M to these cells produced VI modulation and reduced the amount of current inhibited via the VD pathway. Together, these results confirm the requirement for Gbetagamma in VD modulation and implicate Galpha(q)-GTP and Gbetagamma as components in the potentially novel VI pathway.

Multiple G-protein betagamma combinations produce voltage-dependent inhibition of N-type calcium channels in rat superior cervical ganglion neurons.

Activation of several G-protein-coupled receptors leads to voltage-dependent (VD) inhibition of N- and P/Q-type Ca(2+) channels via G-protein betagamma subunits (Gbetagamma). The purpose of the present study was to determine the ability of different Gbetagamma combinations to produce VD inhibition of N-type Ca(2+) channels in rat superior cervical ganglion neurons. Various Gbetagamma combinations were heterologously overexpressed by intranuclear microinjection of cDNA and tonic VD Ca(2+) channel inhibition evaluated using the whole-cell voltage-clamp technique. Overexpression of Gbeta1-Gbeta5, in combination with several different Ggamma subunits, resulted in tonic VD Ca(2+) channel inhibition. Robust Ca(2+) channel modulation required coexpression of both Gbeta and Ggamma. Expression of either subunit alone produced minimal effects. To substantiate the apparent lack of Gbetagamma specificity, we examined whether heterologously expressed Gbetagamma displaced native Gbetagamma from heterotrimeric complexes. To this end, mutant Gbeta subunits were constructed that differentially modulated N-type Ca(2+) and G-protein-gated inward rectifier K(+) channels. Results from these studies indicated that significant displacement does not occur, and thus the observed Gbetagamma modulation can be attributed directly to the heterologously expressed Gbetagamma combinations.

Heterologous expression of a green fluorescent protein-pertussis toxin S1 subunit fusion construct disrupts calcium channel modulation in rat superior cervical ganglion neurons.

The fusion construct pEGFP-PTXS1 was assembled by ligating cDNA encoding the S1 subunit of Bordetella pertussis toxin (PTX) into the plasmid pEGFP-C1 (which codes for enhanced green fluorescent protein). Microinjection of pEGFP-PTXS1 (1-100 ng/microl) into the nucleus of dissociated rat sympathetic ganglion neurons resulted in functional expression as determined from the diffuse green fluorescence and disruption of norepinephrine-mediated N-type Ca2+ channel modulation. The heterologously expressed toxin retained specificity for G alpha(i/o)-dependent pathways as VIP-mediated modulation of N-type Ca2+ channels and muscarine-mediated inhibition of M-type K+ channels persisted in pEGFP-PTXS1 expressing neurons. These data demonstrate that the S1 subunit of PTX is readily expressed in mammalian neurons and remains functional following fusion to the C-terminus of another protein.

Heterologous expression and coupling of G protein-gated inwardly rectifying K+ channels in adult rat sympathetic neurons.

1. G protein-gated inwardly rectifying K+ (GIRK) channels were heterologously expressed in rat superior cervical ganglion (SCG) neurons by intranuclear microinjection. The properties of GIRK channels and their coupling to native receptors were characterized using the whole-cell patch-clamp technique. 2. Following coinjection of either GIRK1-2 or GIRK1-4 cDNA, application of noradrenaline (NA) produced large inwardly rectifying K+ currents. Injection of cDNA encoding individual GIRK subunits produced only small and inconsistent NA-activated inward currents. Current arising from the native expression of GIRK channels in SCG neurons was not observed. 3. NA-mediated activation of GIRK channels was abolished by pertussis toxin (PTX) pretreatment, indicating coupling via G proteins of the Gi/Go subfamily. Conversely, vasoactive intestinal peptide (VIP) activated GIRK channel currents via a cholera toxin-sensitive pathway suggesting coupling through Galphas. Pretreatment of neurons with PTX caused a significant increase in amplitude of the VIP-mediated GIRK channel currents when compared with untreated cells. 4. Application of adenosine, prostaglandin E2 and somatostatin resulted in activation of GIRK channel currents. Activation of m1 muscarinic acetylcholine receptors (i.e. application of oxotremorine M to PTX-treated neurons) failed to elicit overt GIRK channel currents. 5. GIRK channel overexpression decreased basal Ca2+ channel facilitation significantly when compared with uninjected neurons. Furthermore, the NA-mediated inhibition of Ca2+ channels was significantly attenuated. 6. In summary, the ability to heterologously express GIRK channels in adult sympathetic neurons allows the experimental alteration of receptor-G protein-effector stoichiometry. Such studies may increase our understanding of the mechanisms underlying ion channel modulation by G proteins in a neuronal environment.

Modulation of Ca2+ channels by cyclic nucleotide cross activation of opposing protein kinases in rabbit portal vein.

Cyclic nucleotides are known to modify voltage-gated (L-type) Ca2+ channel activity in vascular smooth muscle cells, but the exact mechanism(s) underlying these effects is not well defined. The purpose of the present study was to investigate the modulatory role of the cAMP- and cGMP-dependent protein kinase (PKA and PKG, respectively) pathways in Ca2+ channel function by using both conventional and perforated-patch-clamp techniques in rabbit portal vein myocytes. The membrane-permeable cAMP derivative, 8-bromo cAMP (0.1 to 10 micromol/L), significantly increased (14% to 16%) peak Ba2+ currents, whereas higher concentrations (0.05 to 0.1 mmol/L) decreased Ba2+ currents (23% to 31%). In contrast, 8-bromo cGMP inhibited Ba2+ currents at all concentrations tested (0.01 to 1 mmol/L). Basal Ca2+ channel currents were significantly inhibited by the PKA blocker 8-Bromo-2'-O-monobutyryladenosine-3',5'-monophosphorothioate, Rp-isomer (Rp 8-Br-MP cAMPS, 30 micromol/L) and enhanced by the PKG inhibitor beta-Phenyl-1,N2-etheno-8-bromoguanosine-3',5'-monophosphorothioate, Rp-isomer (Rp-8-Br PET cGMPS, 10 nmol/L). In the presence of Rp 8-bromo PET cGMPS (10 to 100 nmol/L), both 8-bromo cAMP (0.1 mmol/L) and 8-bromo cGMP (0.1 mmol/L) enhanced Ba2+ currents (13% to 39%). The excitatory effect of 8-bromo cGMP was blocked by Rp 8-bromo MB-cAMPS. Both 8-bromo cAMP (0.05 mmol/L) and forskolin (10 micromol/L) elicited time-dependent effects, including an initial enhancement followed by suppression of Ba2+ currents. Ba2+ currents were also enhanced when cells were dialyzed with the catalytic subunit of PKA. This effect was reversed by the PKA blocker KT 5720 (200 nmol/L). Our results suggest that cAMP/PKA stimulation enhances and cGMP/PKG stimulation inhibits L-type Ca2+ channel activity in rabbit portal vein myocytes. Our results further suggest that both cAMP and cGMP have a primary action mediated by their own kinase as well as a secondary action mediated by the opposing kinase.

Goserelin followed by assisted reproduction: results in infertile women with endometriosis.

OBJECTIVE: Demonstrate the usefulness of combined treatment of a Gn-RH agonist and assisted reproduction in the management of infertile women with endometriosis. DESIGN: A prospective evaluation of goserelin's action (Gn-RH(a)) in the extension of endometriosis, of its suppression and clinical improvement, and in pregnancy rates after an immediate assisted reproduction program. SITE: Infertility clinic at a private hospital related to other university hospitals. PATIENTS: 18 infertile patients with laparoscopically-confirmed endometriosis. METHOD: All women were submitted to general laboratory tests, FSH, LH and estradiol measurements, and laparoscopy before and after treatment. All were treated for 6 months with goserelin and, when menstruating, the patients were submitted to an assisted reproduction program with a scheme of HMG + FSH + HCG. MAIN OUTCOME MEASURES: Improvement of endometriosis and achievement of pregnancy. RESULTS: An improvement of the endometriosis score was confirmed in 100% of the cases. The average pretreatment score of 44.8 points decreased to 18.3 after treatment. Similarly, the pain reported by eight of the patients practically disappeared after using the Gn-RH analogue. During treatment with goserelin, all women had amenorrhea. Their periods resumed in an average of 80.5 days after the last injection. In three (17.6%) cases, no follicular response was obtained, and stimulation was suspended. The remaining responses were good: eight GIFT procedures, four IVF-ET procedures and two IUIs resulted in eight pregnancies (57.1%), one of which terminated in an abortion (the patient became pregnant again). The eight pregnancies had good results: one was double and another quadruple. Most importantly, all pregnancies were achieved during the first treatment attempt. CONCLUSION: Combined treatment of goserelin with immediate assisted reproduction is a satisfactory procedure, which achieves a high percentage of pregnancies at the first try and with few abortions in cases of infertile women with endometriosis.

Endometrial pathology and infertility.

OBJECTIVE: To call attention to endometrial pathologies, which in addition to causing menstrual problems, are a cause of infertility. DESIGN: Controlled clinical study. SETTING: Specialized unit in the management of infertile patients. PATIENT(S): Fifteen infertile women between the ages of 26 and 39 years and suffering infertility from endometrial problems for a period of 4 to 18 years were included in the study. Six patients had primary infertility and nine others had secondary infertility. INTERVENTION(S): Once the endometrial pathology was diagnosed, treatment was initiated according to the type of problem: hysteroscopy, curettage, and hormonal replacement with or without corticoids or antiphymic drugs. MAIN OUTCOME MEASURE(S): Clinical studies, laboratory tests, hormonal serum levels, and endoscopy. RESULT(S): After initiating specific treatment for each of the pathologies, menstruation was re-established in 14 of 15 patients. Nine patients became pregnant (8 of 10 cases with bone, squamous cell, or muscular metaplasia). CONCLUSION(S): Pathological changes of the endometrium are causes of infertility. These problems are not as rare as thought. They must be searched for carefully and diagnosed promptly. The majority carry a good prognosis when adequately treated.

Dihydropyridine-sensitive Ca2+ influx modulated by stretch in A7r5 vascular smooth muscle cells.

We examined 45Ca2+ influx in A7r5 vascular smooth muscle cells under cyclical stretch and static conditions and compared the results obtained at resting membrane potential (2.5 mM [K+]o, Em = -58 mV according to uptake of [3H]tetraphenylphosphonium) with those under depolarizing conditions (70 mM [K+]o, Em = -27 mV). Application of 10% average strain (24% maximum) in cycles of 3 s on, 3 s off at resting Em caused a 5-fold increase in Ca2+ influx rate to a level similar to depolarized cells and depolarized, stretched cells. 1 mu M (+)-isradipine blocked 90% of the stretch- or depolarization-activated Ca2+ uptake. When the cells were stretched under Na+ -free conditions, a reduction, not activation, of Ca2+ influx rate occurred. Our results suggest that stretching of cultured aortic vascular smooth muscle cells enhances Ca2+ uptake through a voltage-dependent, dihydropyridine-sensitive Ca2+ entry pathway, whose activation by stretch is dependent upon extracellular Na+.

Modulation of dihydropyridine receptors in vascular smooth muscle cells by membrane potential and cell proliferation.

We have studied binding of isradipine to A7r5 vascular smooth muscle cells as a function of membrane potential and cell proliferation. Consistent with a voltage-modulated receptor model, two classes of binding sites were detected in confluent cultures: high-affinity sites under depolarizing (50 mM K+) conditions (Kd = 45 +/- 3 pM), and lower affinity sites under resting (5 mM K+) conditions (Kd = 181 +/- 20 pM). However, proliferating cells also displayed the high-affinity state at rest (Kd = 29 +/- 9 pM) in addition to a low-affinity site (Kd = 869 +/- 383 pM). Analysis of dissociation rates also revealed two receptor classes during proliferation. Proliferating cells showed a single class of high-affinity sites (Kd = 39 +/- 6 pM) when depolarized, similar to confluent cells. Receptor density in confluent monolayers increased from 15 +/- 3 fmol/10(6) cells at 5 days to 72 +/- 6 fmol/10(6) cells after 10 days. These results suggest (i) that some L-type Ca2+ channels are spontaneously active in proliferating vascular smooth muscle cells, but require depolarization to activate in a confluent monolayer, and (ii) that the density of dihydropyridine receptors increases after a monolayer becomes confluent.

Acute intramuscular injection of oils or the oleic acid component protects mice against paraquat lethality.

Although selenium or vitamin E deficiencies or changing from cereal-based to purified diets augments paraquat toxicity, the action of other dietary components in normal animals fed nutritionally adequate diets is not clear. Upon injection of mice with antiinflammatory agents, a protective action of the corn oil vehicle against paraquat lethalities was noted. This preventive action of a large parenteral administration of unknown components in oils served as the basis for this study. Intramuscular injection of various vegetable oils protected similarly, indicating that in mixtures, the degree of lipid saturation did not seem to be an important factor. Injection of the monounsaturated fatty acid oleic acid decreased oral paraquat lethalities in mice, but linoleic, gamma-linoleic or linolenic acids were not protective in either male or female mice. Measurement of paraquat concentrations in various tissues at various times after administration indicated no effect of corn oil on paraquat distribution. Although the exact mechanism of the complex nature of oil protection against paraquat toxicity in mice is still unknown, this study provides evidence for in vivo oxidant protection by a monounsaturated fatty acid.

Comparative efficacy of gestrinone and danazol in infertile women with endometriosis.

The relationship between endometriosis and infertility is observed frequently. Patients with both conditions require a conservative approach to their management. Since hormonal therapy is one of those approaches, we sought to compare the efficacy of Danazol and Gestrinone in 80 infertile patients (48 and 32, respectively). Therapy lasted 6 months in both treatment groups, and all patients studied had laboratory tests performed and were clinically evaluated and classified through laparoscopy before and after therapy. The improvement of symptoms and favorable follow-up were similar with both treatments. The reestablishment of menstrual patterns and fertility were also nearly alike in both groups. However, Gestrinone was associated with fewer secondary effects and is easier to administer than Danazol. We conclude that Gestrinone is a useful medication in the management of the infertile patient with endometriosis.

Hyperprolactinemia and mammary prostheses. A report of eight cases.

Eight young women developed clinical manifestations of hyperprolactinemia after the application of bilateral methylpolixolosane mammary prostheses. Elevated serum levels of prolactin were present in all cases. Treatment with bromocriptine or methergoline corrected the excessive prolactin production and associated symptoms. The pathophysiology of this syndrome is unexplained as yet.

Utilidad de bromocriptina en la esterilidad anovulatoria con prolactina normal / Utility of bromocriptine in the anovulatory sterility with normal prolactin

Se estudió a 90 pacientes del Centro para el Estudio de la Fertilidad, con objeto de valorar la utilidad de la administración de bromocriptina en mujeres sanas estériles con trastornos anovulatorios y niveles normales de prolactina. A cada paciente se realizaron tres determinaciones de prolactina, y de acuerdo con estos resultados se clasificaron en cinco grupos. Asimismo se efectuó un ciclo de control con determinaciones hormonales, registro seriado de moco cervical, ultrasonografía diaria de crecimiento folicular y calendario coital o programación de inseminaciones. El tratamiento consistió en administración de bromocriptina en dosis de 2.5 mg dos veces al día. Los resultados fueron: ovulación en 86.84% de pacientes con niveles normales de prolactina, y embarazo en 44.74%. Además, se restituyó la ovulación y se obtuvo embarazo más temprano en los casos con niveles normales de prolactina que en las pacientes que presentaban hiperprolactinemia. Se concluye que la administración de bromocriptina como tratamiento de alteraciones de tipo anovulatorio en pacientes con niveles normales de prolactina, es una arma terapéutica eficaz, en especial en los casos en los que otros inductores de la ovulación, aparentemente adecuados, han fracasado

Pregnancy in hyperprolactinemic women.

Pregnancy achieved in women who receive treatment to correct the secretory dysfunction of nontumoral HPRL or microprolactinomas requires close prenatal care, but generally its course does not vary from normal. When a macroprolactinoma is present, consequences of pregnancy are insignificant, provided the tumor has been previously treated or bromocriptine is given continuously during the pregnancy. On those rare occasions when symptoms of tumor growth appear during pregnancy, bromocriptine and dexamethasone effectively control such manifestations. Breast-feeding of the infant can be allowed, and a second pregnancy within a short term is not contraindicated. When a new pregnancy is not desired, nonhormonal contraceptive methods are advised. Patients with nontumoral HPRL and microadenomas require periodic checkups. Macroadenomas may be surgically excised, but longterm bromocriptine treatment also achieves good results and is highly recommended.

Cesarean section IUD insertion.

A trial was carried out on 154 selected volunteers who underwent cesarean section and then received an ML Cu 250 IUD during the operation. 56% of the operations were planned while 44% were not. The IUD was inserted in 65, 80, and 9 cases in the 1st, 2nd, and 3rd cesarean section, respectively. The patients were followed-up after 1, 3, 6, 12, and 24 months. Hospitalization time and postoperative morbidity did not increase with device insertion. Maternal lactation was not altered. 8% of the patients had postpartum bleeding which lasted more than 40 days. At the 1 year examination, 52% of the patients were menstruating normally while at 2 years, the percentage had increased to 62.5%. The acceptability, continuity, and efficiency of the contraceptive procedure proved successful. There were only 4 cases (2.6%) in which the device had to be removed for medical reasons (due largely to endometritis) and 8 cases (5.2%) because of patient desire (menstrual abnormalities). Spontaneous expulsion of the IUD occurred in 4 cases (2.6%). As of the present time, no pregnancies have been detected. The cesarean section insertion of an ML Cu 250 IUD produced excellent results and further, more widespread use is suggested.