Progesterone modulation of D5 receptor expression in hypothalamic ANP neurons, the role of estrogen.

Aberrant responses to dopamine consequent to a reduction of D1-like receptors in critical regions of the brain have been implicated in schizophrenia. Whereas estrogen may protect against the onset and incidence of psychosis in the illness, the neurobiological effects of the ovarian steroid remain unclear. Recently we have shown that estrogen augments the expression of D5 receptors, a member of the D1-like receptor family, in central neurons and enhances the functions of the host cells. Employing rat hypothalamic neuron cultures, we report here that another important ovarian steroid, progesterone, also augments dopamine D5 receptor expression in hypothalamic atrial natriuretic factor (ANP) neurons. However, unlike the effect of estrogen, progesterone acts indirectly through potentiating estrogen-mediated changes that include enhancement of D5 receptor expression, immunoreactive (ir)-ANP release and pro-ANP mRNA abundance. We conclude that whilst progesterone has little effect by itself, the steroid works in synergism with estrogen to augment the function of ANP neurons. The possibility that progesterone may further enhance the protective effect of estrogen against the incidence of psychosis in schizophrenia now needs to be considered.

Automated static perimetry: the influence of myopia and its method of correction.

OBJECTIVE: To determine how the magnitude of myopia and its method of correction influence visual field testing. DESIGN: Prospective observational case series, including comparison of spectacles and contact lenses. PARTICIPANTS: One hundred forty-six ophthalmologically normal males 19 to 24 years of age with myopia (spherical equivalent from -0.50 to -14.0 diopters). METHODS: Participants performed automated static threshold perimetry. Refractive errors were corrected using trial lenses and soft contact lenses. Subjects were tested with both methods of correction, the order of which was randomized. MAIN OUTCOME MEASURES: Threshold sensitivity and global indexes. RESULTS: Only one subject (0.7%) had a significant reproducible visual field defect on both methods of correction. Six subjects (4.1%) had a focal visual field defect on one method of correction but not the other. For myopia greater than -4.0 diopters, the mean defect decreased significantly as axial length and degree of myopia increased (P: < 0.01). Similar results were obtained with either method of correction. CONCLUSIONS: Threshold sensitivity is reduced in moderate and high myopia, regardless of the method of correction. The surprisingly low prevalence of visual field defects in this myopic population disputes the widely held view that myopia is associated commonly with visual field abnormalities. If field defects are found in myopes on automated perimetry, it is advisable to repeat the test with another method of optical correction to check that such defects are genuine and not related to the method of refractive correction.

Glucocorticoid modulation of dopamine mediated effects on hypothalamic atrial natriuretic factor neurons.

Dopamine (DA) plays an important role in cognition, neuroendocrine functions and psychosis.1,2 Whilst stress adversely affects some of these functions, its neurobiological basis remains unclear.3 In the rat hypothalamus, a concurrent activation of D5and D2 receptors by dopamine produces a biphasic effect on the function of atrial natriuretic factor (ANF) neurons.4 Whereas low doses (10-8 and 10-7 M) of DA suppress the release and pro-ANF mRNA expression, high doses (10-6 and 10-5 M) of the amine produce an opposite effect through the interaction of D5 and D2 receptors. We report here that the augmenting effect of DA on the hypothalamic neurons is inhibited by a synthetic glucocorticoid, dexamethasone (DM), in both time-dependent and dose-related manner with an EC50 of 0.1 nM. Furthermore, the inhibition is blocked by 100 nM of RU38486 (P<0.01), a glucocorticoid receptor antagonist, but not by an equivalent dose of RU28318, a mineralocorticoid receptor antagonist. In contrast, DM failed to modulate low doses (10(-8) to 10(-7) M) of DA-induced suppression of ir-ANF release and pro-ANF mRNA expression that was mediated primarily through D2 receptors. We conclude that glucocorticoids markedly alter DA-induced biphasic effects by down-regulating D5, but not D2, receptor-mediated neurobiological events. Hence, in severe stress, high levels of circulating glucocorticoids may render dopamine to act as a potent suppressor of neurons that possess both D5 and D2 receptors. The possibility that this novel mechanism of stress hormone or glucocorticoids may, in part, undermine DA-mediated neurophysiology in critical regions of the brain, which links to psychosis now needs to be considered.

Relative abundance of placental pro-atrial natriuretic factor mRNA in normal pregnancy and pre-eclampsia.

Atrial natriuretic factor (ANF), produced by cytotrophoblast cells of the human placenta, may be involved in the regulation of uteroplacental blood flow. Pre-eclampsia is associated with maternal hypertension and reduced uteroplacental perfusion. The relationship between pre-eclampsia and placental production of ANF is not known. This study measured pro-ANF mRNA levels by Northern blot analysis in placentae delivered by caesarean section at preterm and term gestations from women with normotensive and pre-eclamptic pregnancies and found no significant difference between pre-eclampsia and normal pregnancy at either gestation. This result suggests that placental production of ANF is not altered at the pretranslational level during pre-eclampsia.

Dopamine induces a biphasic modulation of hypothalamic ANF neurons: a ligand concentration-dependent effect involving D5 and D2 receptor interaction.

Increasing evidence now suggests that more than one subtype of dopamine receptors is co-expressed in some of the central neurons. The neurobiological effects on the host cells when these receptors are concurrently activated by their common physiological ligand, dopamine, however, remains elusive. Among the members of the family of dopamine receptors, coupling of D1-like dopamine receptors to Gs and D2-like receptors to Gi proteins are known to augment or suppress cellular functions respectively, through modulation of adenylyl cyclase activity and consequently cAMP generation. Simultaneous activation of D1 and D2 receptors in transfected cell lines expressing the two cloned receptors, however, produced antagonistic effects. This is in contrast to in vivo studies, in which concurrent activation of D1-like and D2-like receptors by their respective agonists may induce synergistic or antagonistic effects or both. We report here that in long-term rat hypothalamic cell cultures, activation of both D1-like (D1 and D5) and D2 receptors on atrial natriuretic factor-producing neurons by dopamine yields a biphasic response. The response is ligand concentration-dependent and involves type II adenylyl cyclases. This process is mediated primarily through antagonistic and synergistic interactions of D5 and D2 receptors as the event is mimicked by the concurrent activation of these two receptors co-transfected in CHO cells. Our present findings suggest a novel action of dopamine, and the biochemical processes involved may underlie some of the pharmacological actions of atypical anti-psychotic drugs. Molecular Psychiatry (2000) 5, 39-48.

Endogenous Klebsiella endophthalmitis.

PURPOSE: To report 4 cases of endogenous Klebsiella endophthalmitis. This disease is a rare but devastating complication of Klebsiella sepsis. Familiarity with its varied clinical presentations would aid in early diagnosis and institution of appropriate therapy. METHODS: We report 5 eyes of 4 patients with endogenous Klebsiella endophthalmitis from December 1995 to September 1996 and review the literature regarding previously successfully treated cases. RESULTS: The mean age of the patients was 67.5 years (range 37-85 years). One patient had bilateral involvement. At presentation, 3 eyes had a visual acuity of no perception of light (NPL) and the 1 patient with bilateral involvement had an acuity of counting fingers in one eye and 20/40 in the other. The primary source of sepsis was the pulmonary system in 2 patients, the hepatobiliary system in 1 patient and the urinary tract in 1 patient. One patient had coexisting diabetes mellitus. Two patients had a pupillary hypopyon. Local and systemic treatment with an aminoglycoside and cephalosporin was initiated. Despite treatment, 2 eyes remained NPL. One eye developed a globe perforation and was eviscerated. The patient with bilateral involvement was treated within 8 h and recovered fully with a final visual acuity of 20/20 in both eyes. CONCLUSION: It is important to have a high index of suspicion in patients with septicaemia presenting with intraocular inflammation, especially in diabetics with suppurative liver disease, urinary tract infection or pulmonary infection. As the visual prognosis for patients with Klebsiella endophthalmitis is generally poor, early diagnosis and treatment with intravenous and intravitreal antibiotics may improve the visual outcome.

D5 dopamine receptors mediate estrogen-induced stimulation of hypothalamic atrial natriuretic factor neurons.

Whereas progesterone and dopamine share a common central pathway to modulate sexual behavior in female rats, the way in which estrogen is involved remains unclear. In a long-term rat hypothalamic cell culture system, atrial natriuretic factor-producing neurons were identified as candidate sites for integration of sex steroid action. Estrogen induces the expression of progesterone receptors in atrial natriuretic factor neurons and also augments neuronal functions by increasing expression of constitutively active D5 receptors that generate cAMP in a ligand-independent manner. Such a cross-talk mechanism allows estrogen to exert its effects via the adenylyl cyclase-cAMP system by augmenting dopamine receptor activity, an action that may play an important integrative role in facilitating female sexual behavior.

Subjective visual experience during extracapsular cataract extraction and intraocular lens implantation under retrobulbar anaesthesia.

PURPOSE: To investigate the subjective visual experience of patients during cataract surgery under retrobulbar anaesthesia. METHODS: One hundred cataract patients who underwent routine extracapsular cataract extraction and intraocular lens implantation under retrobulbar anaesthesia were interviewed by the authors on the same day after their operation regarding their visual experience in the operated eye during surgery. RESULTS: Forty-three men and 57 women were included in the study. Their mean age was 68.4 years (range 40-87 years). Their pre-operative best corrected visual acuity ranged from 6/12 to perception of light. Eighty-four eyes had no other ocular pathology apart from cataract. Twenty patients reported no light perception during the surgery. The rest reported that they could see light (80 patients), one or more colours (56 patients), movements (39 patients), flashes (36 patients), instruments (16 patients) and/or the surgeon's fingers or hands (10 patients). The colours seen included red (29 patients), yellow (29 patients), green (12 patients), blue (11 patients) and orange (2 patients). Fifteen patients saw a spectrum of colours similar to that of the rainbow. Forty-four patients reported that the brightness of light changed during the operation. Five patients found their visual experience frightening. There was no correlation between those who found the experience frightening and the sex or age of patient, presence of coexisting ocular pathology, duration of operation, whether the operation was the first or second cataract operation in the patient, or the type of visual sensation experienced. CONCLUSION: Many patients undergoing cataract surgery under retrobulbar anaesthesia experience a variety of visual sensations that may be frightening in a small proportion of cases.

The ICS-'BPH' Study: uroflowmetry, lower urinary tract symptoms and bladder outlet obstruction.

OBJECTIVE: To explore the relationship between uroflow variables and lower urinary tract symptoms (LUTS): to define performance statistics (sensitivity, specificity, positive and negative predictive values) for maximum urinary flow rate (Qmax) with respect to bladder outlet obstruction (BOO) at various threshold values; and to investigate the diagnostic value of low-volume voids. PATIENTS AND METHODS: The study comprised 1271 men aged between 45 and 88 years recruited from 12 centres in Europe, Australia, Canada, Taiwan and Japan over a 2-year period. Symptom questionnaires, voiding diaries, uroflowmetry and pressure-flow data were recorded. The relationship between uroflow variables and symptoms, Qmax and BOO, and the diagnostic performance of low volume voids were analysed. RESULTS: The relationship between symptoms and uroflow variables was poor. The mean difference between home-recorded and clinic-recorded voided volumes was -48 mL. Qmax was significantly lower in those with BOO (9.7 mL/s for void 1) than in those with no obstruction (12.6mL/s; P<0.001) and Qmax was negatively correlated with obstruction grade (Spearman's correlation coefficient -0.3, P<0.001), even when controlling for the negative correlation between age and Qmax (Spearman's partial correlation coefficient -0.29, P<0.001). A threshold value of Qmax of 10 mL/s had a specificity of 70%, a positive predictive value (PPV) of 70% and a sensitivity of 47% for BOO. The specificity using a threshold Qmax of 15 mL/s was 38%, the PPV 67% and the sensitivity 82%. Those voiding <150 mL (n=225) had a 72% chance of BOO (overall prevalence of BOO 60%). In those voiding >150 mL the likelihood of BOO was 56%. The addition of a specific threshold of 10 mL/s to these higher volume voiders improved the PPV for BOO to 69%. CONCLUSION: While uroflowmetry cannot replace pressure-flow studies in the diagnosis of BOO. it can provide a valuable improvement over symptoms alone in the diagnosis of the cause of lower urinary tract dysfunction in men presenting with LUTS. This study provides performance statistics for Qmax with respect to BOO: such statistics may be used to define more accurately the presence or absence of BOO in men presenting with LUTS, so avoiding the need for formal pressure-flow studies in everyday clinical practice, while improving the likelihood of a successful outcome from prostatectomy. This study also shows that low-volume uroflowmetry can provide useful diagnostic information and that, as such, the data from such voids should not be discarded.

An outbreak of cutaneous aspergillosis in a tertiary-care hospital.

A cluster of four cases of surgical and burn wound aspergillosis occurred in a 900-bed, adult, tertiary-care hospital. The source was traced to the outside packages of dressing supplies, which had become contaminated during construction in the central Inventory Control area. This resulted in patients with large exposed surface areas being inoculated directly with Aspergillus spores.

Glucocorticoid stimulation of pro-ANF mRNA expression of adult rat hypothalamic neurons in culture: a cAMP-dependent effect.

We describe here long term primary cultures of adult rat hypothalamic cells, a novel system which we have exploited to examine the effect of glucocorticoids on adult ANF neurons in vitro. ANF neurons with and without neurite outgrowths were identified by immunocytochemistry and by in situ hybridization studies, in which pro-ANF mRNA signals were localised in cultured cells. Dexamethasone (DM) augmented the abundance of pro-ANF mRNA signals in a dose-related manner with ED50 of 5 x 10(-12)M and Emax of 10(-10)M. This stimulation effect was mimicked by corticosterone but not by deoxycorticosterone. Similarly, activation of the adenylyl cyclase-cAMP system with forskolin, 8-brom-cAMP or 3-isobutyl-1-methylxanthine (IBMX), also markedly elevated the level of pro-ANF mRNA abundance. Interestingly, whereas forskolin-induced stimulation was reversed by Rp-cAMP, a cAMP antagonist, the augmentation induced by DM alone or by DM and forskolin together, was similarly suppressed by the chemical. We therefore conclude that (i) adult ANF neurons are capable of regenerating neurite outgrowths in vitro, (ii) cAMP dependent pathways play a fundamental role in regulating the expression of pro-ANF mRNA, and (iii) the stimulatory effect of glucocorticoids is indirect and mediated, at least in part, through modulating the activity of cAMP dependent pathways.

Ascorbic acid augments the adenylyl cyclase-cAMP system mediated POMC mRNA expression and beta-endorphin secretion from hypothalamic neurons in culture.

Besides acting as an important cofactor in the biosynthesis of catecholamine, ascorbic acid (AA) also modulates the activity of peptidylglycine-alpha-amidating monooxygenase for the post-translational modification of neuropeptides such as alpha-MSH and TRH. We report here a novel action of AA in modulating the secretion of immunoreactive beta-endorphin (ir-beta EP) and mRNA expression of proopiomelanocortin (POMC) following the activation of cAMP-dependent protein kinase A pathway in rat hypothalamic neurons. Primary cultures of hypothalamic neurons from neonatal rats as previously described were employed in the present studies. Six days after plating, cultures were replenished with serum-free media and incubated with vehicle or various doses of AA in the presence or absence of forskolin, 3-isobutyl-1-methylxanthine (IBMX), N6,2'-O-dibutyryladenosine 3'5'-(cyclic)monophosphate [(Bu)2cAMP]. Whereas the basal ir-beta EP release was 22.0 +/- 0.4 pg/well (mean +/- S.E.; n = 3), 10 microM of forskolin treatment increased ir-beta EP release approximately 4.2-fold. Co-incubation with AA enhanced forskolin induced ir-beta EP release and that this enhancing effect of AA was both time related and dose-dependent, with an ED50 of approximately 10 microM and an Emax of 100 microM. At the concentration of 10 microM, AA augmented ir-beta EP release approximately 6.1-fold that of cultures treated with forskolin alone. A similar potentiating effect of AA was also seen in cultures co-treated with IBMX or with (Bu)2cAMP. These enhancing effects of AA were similarly found in the abundance of total cAMP and of POMC mRNA of cultures which received identical treatments. However, it is important to point out that AA alone did not modulate ir-beta EP release or the abundance of POMC mRNA or total cAMP levels of the hypothalamic cultures when protein kinase A pathway was not activated. We thus conclude that AA augments cAMP-dependent protein kinase A pathway-induced production and release of beta EP from rat hypothalamic neurons in culture. Furthermore, this biological effect of AA is, at least in part, mediated through enhancing the responsiveness of the adenylyl cyclase-cAMP system.

Glucocorticoids inhibit D1B, but not D2, receptor-mediated effects on hypothalamic atrial natriuretic factor neurons.

Recent evidence suggests that ANF neurons of the hypothalamus are dopamine sensitive, and the catecholamine may exert a direct stimulatory or inhibitory effect on the neurons mediated through D1 or D2 receptors, respectively, in a manner related to the differential dopamine binding sensitivity of the two receptor subtypes. Employing well characterized ANF RIA and colorimetric Northern blot analysis with synthetic oligonucleotide probes complementary to pro-ANF messenger RNA (mRNA), we report here the effect of dexamethasone (DM), a potent synthetic glucocorticoid, on DA-stimulated ANF neurons in long term primary cultures of neonatal rat hypothalamic cells. Although DM alone did not affect basal secretion of immunoreactive ANF, it approximately halved immunoreactive ANF secretion induced by D1 agonist, SKF38393 (P < 0.01). The effect of DM was both time dependent and dose related, with an EC50 of 0.1 nM; it was blocked by 100 nM RU38486 (P < 0.05), a glucocorticoid receptor antagonist, but not by 100 nM RU28318, a mineralocorticoid receptor antagonist. In addition, the effect of DM was mimicked by corticosterone (EC50, 10 nM), but not deoxycorticosterone. The increased expression of pro-ANF mRNA signal induced by the D1 agonist in culture was suppressed by DM in a similar manner. In contrast, DM did not modulate ANF production and secretion induced by D2 agonist, quinpirole. Furthermore, reverse transcription-polymerase chain reaction demonstrated that D1B, but not D2, receptor mRNA expression was selectively suppressed by glucocorticoids. Thus, we conclude that in monolayer cultures of rat hypothalamic neurons, glucocorticoids differentially modulate dopamine receptor-induced responsiveness of ANF neurons by down-regulating D1B, but not D2, receptor-mediated changes. Hence, in severe stress, high levels of circulating glucocorticoids may negate the D1B-induced stimulatory response but allow dopamine to suppress the function of hypothalamic ANF neurons through D2 receptor activation.

Atrial natriuretic factor production by the human placenta.

Atrial natriuretic factor (ANF), a 28 amino acid peptide, is primarily produced and secreted by cardiac atrial myocytes to modulate cardiovascular and renal functions. Although ANF is also produced in tissues other than the heart, it remains uncertain whether the peptide is synthesised by human placentae. We provide here evidence from in vitro studies suggesting de novo production of ANF by the human placenta. Placental tissues were collected from normal term pregnancies by elective Cesarean section and acid extracted for ANF radioimmunoassay. The level of placental immunoreactive (ir) ANF was 186 +/- 33 pg/gm wet tissues (mean +/- SE, n = 6). Perfusates from in vitro perfusion of the fetoplacental compartment of placental lobules yielded 26 +/- 6 pg/min/gm wet tissue (n = 3) of irANF. Furthermore, pro-ANF mRNA signals were localised by colorimetric in situ hybridization in a subpopulation of placental cytotrophoblast-like cells, but not in syncytiotrophoblasts nor in chorionic cells of placental sections. Northern blot analysis of placental tissue extracts showed a single band of pro-ANF mRNA signals (-0.85 Kb) similar in size to that found in the rat heart. Our findings suggest that ANF is expressed and produced by a small population of human placental cytotrophoblast-like cells. The possibility that placental ANF may be secreted locally or into the fetoplacental circulation to exert paracrine, autocrine or both effects now needs to be considered.

Glutamate enhances the adenylyl cyclase-cAMP system-induced beta-endorphin secretion and POMC mRNA expression in rat hypothalamic neurons in culture: NMDA receptor-mediated modulation.

L-Glutamate, a major excitatory amino acid of the central nervous system, plays important roles as neurotransmitter and neuromodulator in the brain. Increasing evidence suggests that glutamate may also involve in the regulation of the neuroendocrine system at the hypothalamus. Employing long term monolayer hypothalamic cell cultures prepared from neonatal rats, we reported here that whereas glutamate significantly enhanced forskolin-, or N6,2'-O-dibutyryladenosine-3'5'-cyclic monophosphate [(Bu)2cAMP]-stimulated immunoreactive (ir)-beta EP release from cultures treated daily for 4 consecutive days, the excitatory amino acid alone produced little effect. This potentiation of glutamate was time-related and dose-dependent with an Emax value of the amino acid being approximately 50 microM; at this concentration glutamate augmented ir-beta EP secretion about 1.8 times (P < 0.05) that induced by 2 microM forskolin alone. Similar effects were also observed for POMC mRNA levels in cultures subjected to 6 h of the above treatment regime. This potentiating effect of glutamate appears to be mediated specifically through NMDA receptor as it can be mimicked by NMDA but not by kainic acid or quisqualic acid, and blocked by the NMDA receptor antagonist 2-amino-5-phosphonovalerate (APV), but not by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA glutamate receptor antagonist. Interestingly, glutamate was found not to enhance high doses of forskolin (10 microM) or (Bu)2cAMP (100 microM) stimulated beta EP release and POMC mRNA levels in hypothalamic cell cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Progesterone, but not estrogen, modulates the cAMP system mediated ir-beta-endorphin secretion and POMC mRNA expression from rat hypothalamic cells in culture.

It is now evident that hypothalamic beta-endorphin (beta EP) modulates reproductive physiology at the central level by inhibiting the function of neurons producing gonadotropin-releasing hormone (GnRH). Increasing evidence suggests that gonadal steroids, which play an important role in the long-loop negative feedback on the hypothalamus-pituitary-gonadal axis, may exert its indirect inhibitory action through modulating the production and release of hypothalamic beta EP. However, it remains unclear whether progesterone or estrogen alone or their combination is important to exert this effect. Employing long-term monolayer neonatal hypothalamic cell cultures, we reported here that whereas progesterone significantly enhanced forskolin-, N6,2'-O-dibutyryladenosine-3'5'-cyclic monophosphate [(Bu)2cAMP]-, 3-isobutyl-1-methylxanthine (IBMX)- or cholera toxin-stimulated immunoreactive (ir)-beta EP release from cultures treated daily for 4 consecutive days, the steroid alone produced little effect. This potentiation of progesterone was time-related and dose-dependent with an EC50 value of the steroid being approximately 25 nM; at this concentration the steroid increased ir-beta EP secretion about 1.6 times (P < 0.05) that induced by 5 microM forskolin alone. Similar effects were also observed for POMC mRNA levels in cultures subjected to 6 h of the above treatment regime. This potentiating effect appears specific as it can be mimicked by progestin, a progesterone receptor agonist and blocked by the progesterone receptor antagonist RU38486, but not RU28318, a mineralocorticoid receptor antagonist. Furthermore, beta-estradiol alone failed to exert a significant effect on basal, forskolin-induced or on forskolin and progesterone co-stimulated beta EP release or POMC mRNA levels in hypothalamic cell cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

D1 receptors mediate dopamine stimulation of immunoreactive atrial natriuretic factor (ANF) release and pro-ANF messenger ribonucleic acid expression of rat hypothalamic neurons in culture.

Atrial natriuretic factor (ANF) and its smaller congeners are produced and released from rat brains to regulate the cardiovascular system, drinking behavior, and neurohormone release at the central level. In the hypothalamus, the adenylyl cyclase-cAMP system plays an important role in modulating the function of ANF neurons in the paraventricular nuclei and the periventricular regions that receive rich dopaminergic inputs. We report here a novel observation of a dopamine (DA) D1 agonist, SKF-38393, modulating immunoreactive (ir) ANF and pro-ANF messenger RNA (mRNA) expression in rat hypothalamic neurons. In long term primary cultures of neonatal rat hypothalamic cells, treatment with SKF-38393 increased irANF secretion in a time-related and dose-dependent manner, with an ED50 of approximately 10(-7) M and a concentration producing maximum effect of 10(-5) M. This stimulating effect of SKF-38393 was mimicked by 10(-5) M DA, a physiological ligand for D1 receptors. Furthermore, both of the stimulatory effects were abolished by SCH-23390, a D1 antagonist. These immunoassay findings were accompanied by corresponding changes in the abundance of pro-ANF mRNA in the cultures, as examined by colorimetric Northern blot analysis. By combining the techniques of in situ hybridization and immunocytochemistry, the mRNA of D1 receptor was colocalized in approximately 90% of the irANF-positive cells in the cultures. In addition, cholera toxin, an irreversible activator of adenylyl cyclases, markedly increased irANF secretion and cAMP production in a dose-dependent manner. This effect mimicked that of D1 agonist-stimulated ANF release, in that the latter also concurrently enhanced cAMP production in the hypothalamic cultures. Furthermore, the stimulatory effect of D1 agonist on irANF release was markedly suppressed by rp-cAMPS, a cAMP antagonist. We, thus, conclude that the release and gene expression of ANF in rat hypothalamic neurons are directly stimulated by DA acting through its D1 receptors on ANF neurons; this effect may operate at the genomic level and is mediated at least in part through activation of the cAMP-dependent kinase-A pathway.

D2 receptors mediate dopamine suppression of irANF release and pro-ANF mRNA expression of rat hypothalamic neurons in culture.

Although N-terminal truncated forms of atrial natriuretic factor (ANF) are produced and released from rat hypothalamic neurons, the intrahypothalamic regulation of these processes remains unclear. Employing a well-characterized hypothalamic cell culture system, we report here that dopamine, mediating through D2 receptors, inhibits the synthesis and release of ANF. In long-term cultures of hypothalamic neurons, daily treatment for 4 days with quinpirole, a D2 agonist, significantly suppressed the basal irANF release in a time-related and a dose-dependent manner. The ED50 and Emax of the drugs were 9.1 x 10(-8) M and 10(-5) M, respectively. This effect of quinpirole was mimicked by 10(-7) M of dopamine, a physiological ligand for D2 receptor. Furthermore, the suppressing effects of both quinpirole and dopamine were abolished by sulpiride, a D2 antagonist. Whereas 10(-6) M of forskolin treatment consistently enhanced the release of irANF through activating the adenylyl cyclase-cAMP system, this stimulatory effect was suppressed by quinpirole in a dose-related manner. In addition, the application of pertussis toxin, a bacterial toxin which inactivated G1 protein activity, reversed the suppressing effect of quinpirole or dopamine on irANF release. These immunoassay findings were accompanied by corresponding changes in the abundance of pro-ANF mRNA in the cultures as determined by colorimetric Northern blot analysis. By combining the techniques of in situ hybridization and immunocytochemistry, the mRNA of D2 receptor was colocalized with irANF at a single cell level by double fluorescent staining.(ABSTRACT TRUNCATED AT 250 WORDS)

ANF(1-28) is a potent suppressor of pro-opiomelanocortin (POMC) mRNA but a weak inhibitor of beta EP-LI release from AtT-20 cells.

Controversies remain whether atrial natriuretic factor (ANF) may play a role in modulating the release of POMC derived peptides from pituitary corticotrophs. Employing AtT-20 mouse pituitary tumour cells, we report here the effects of rat ANF(1-28) and sodium nitroprusside (SNP), both of which augment cellular levels of cGMP through activating particulate and soluble guanylyl cyclases respectively, on the expression of POMC mRNA abundance. Furthermore, the cellular contents and secretion of (beta endorphin-like immunoreactivity) beta EP-LI from these cultures were also examined. Whereas the abundance of POMC mRNA was found to be markedly suppressed following 4h of incubation with rANP(1-28) (0.01 to 1 microM), SNP (0.1 to 10 microM) and dibutyryl-cGMP (1 to 100 microM) in a dose related manner, only a modest reduction in the release and cell contents of beta EP-LI was found in some of these cultures. It is also of interest to note that in all the cases examined, the inhibitory effect was associated with a significant suppression of cAMP levels in the cultures. Taken together, our present findings suggest that ANF may play a more important role in suppressing the production than the release of POMC related peptides from AtT-20 cells. Thus, it raises the possibility that hypothalamic ANF may likewise modulate the function of the pituitary-adrenal axis through exerting a greater effect on inhibiting the production than the secretion of pituitary ACTH.