Evidence for a role of intracellular-calcium release in nitric oxide-stimulated relaxation of the rabbit corpus cavernosum

We present the clinical findings in a 2 1/2 year old girl with an unusual mosaic karyotype. Amniocentesis was performed at 35 weeks because of intrauterine growth retardation. The in situ cultures showed 47,XX,+15 in seven colonies, 69,XXX in four colonies, and in two colonies 46,XX was detected. Subcultures showed 69,XXX/47,XX,+15 with no normal cells. A small dysmorphic baby was born at term. Cytogenetic studies were performed on cord blood, amnion, and placental tissue immediately after birth and further studies on peripheral blood, bone marrow, muscle biopsy, and skin cultures at 1 1/2 years of age. FISH with two autosomal centromeric probes was performed on the peripheral blood sample. A normal cell line could not be seen in any postnatal tissue by either technique. The predominant cell line postnatally was 69,XXX. There were no cytogenetic polymorphisms and the parental origin of the different cell lines was not determined. Marked red cell macrocytosis of peripheral blood was noted on routine blood count. Bone marrow aspiration showed megaloblastic haemopoiesis without evidence of vitamin B12 or folate deficiency. At 2 1/2 years, the patient has significant developmental problems.     

Dopaminergic regulation of striatal acetylcholine release: the critical role of acetylcholinesterase inhibition

This study examined the effects of different levels of acetylcholinesterase (AChE) inhibition on dopaminergic regulation of striatal acetylcholine (ACh) release as estimated by in vivo brain microdialysis. Systemic administration of d-amphetamine (2 or 10 mg/kg) increased the striatal output of ACh when the AChE inhibitor neostigmine (0.1 microM) was present in the perfusion fluid. In contrast, when the same experiments were conducted at 0.01 microM neostigmine, d-amphetamine failed to affect (2 mg/kg) or significantly decreased (10 mg/kg) striatal ACh output. The inhibitory action of the D2 receptor agonist quinpirole (0.2 mg/kg) was significantly greater at 0.01 microM than at 0.1 microM neostigmine. Similarly, there was a nonsignificant trend for the D2 antagonist raclopride (1 mg/kg) to stimulate ACh release to a greater extent at the low neostigmine concentration. In contrast, the stimulant effects of systemic administration of the D1 agonist A-77636 (1.46 mg/kg) on striatal ACh release were the same at the two neostigmine concentrations. These results demonstrate that the concentration of an AChE inhibitor in the perfusion solution can quantitatively and even qualitatively influence the manner in which dopaminergic agents regulate ACh overflow in the striatum. On comparing the present results with earlier reports concerning the effects of d-amphetamine on tissue concentrations of ACh, it is tentatively concluded that a low neostigmine concentration is the more physiologically relevant condition. Under such conditions, at moderate doses d-amphetamine does not appear to alter striatal ACh release, with this likely being due to the opposing actions of D1 and D2 receptors. Nevertheless, until the endogenous interstitial concentrations of striatal ACh can be measured by other methods, the physiological relevance of ACh microdialysis studies in the striatum will remain uncertain.     

Evidence for a role for the cyclic adenosine 3',5'-monophosphate/protein kinase-A pathway in regulation of the gonadotropin subunit messenger ribonucleic acids

cAMP regulation of gonadotropin secretion and subunit mRNA levels was studied in pituitary cells perifused with pulses of GnRH. Pituitary cells from 7-week-old male rats castrated at 5 weeks of age were stimulated hourly for 9-24 h with 1-min pulses of GnRH, the adenylate cyclase activator forskolin, the cell-permeable cAMP analog 8-bromo-cAMP (8Br-cAMP), or control medium. Cells were also treated with the nonsteroidal antiinflammatory drug flufenamic acid, which reduces pituitary cAMP levels. During perifusion, the effluent was collected in 10-min fractions for FSH and LH assay. At the completion of perifusion, total RNA was extracted, and gonadotropin subunit mRNA levels were quantitated by Northern analysis. Continuous administration of flufenamic acid gradually reduced the amplitude of GnRH-stimulated FSH and LH pulses to nadir values of 40 +/- 4.7% and 62 +/- 12% of the control value, respectively. Flufenamic acid decreased (P < 0.05) FSH beta and alpha-subunit mRNA levels and blocked the effect of GnRH to lengthen LH beta mRNA. Pulses of forskolin or 8Br-cAMP released LH and FSH, and continuous forskolin or 8Br-cAMP potentiated the gonadotropin stimulatory effect of GnRH. Forskolin or 8Br-cAMP increased (P < 0.05) FSH beta mRNA and alpha-subunit mRNA levels when administered in pulses, but not when administered continuously, and lengthened LH beta mRNA. The Nal-Glu GnRH antagonist blocked the effects of GnRH pulses, but not the effects of 8Br-cAMP or forskolin. In conclusion, lowering intracellular cAMP levels with flufenamic acid attenuated GnRH-stimulated gonadotropin secretion, decreased alpha-subunit and FSH beta mRNA levels, and blocked the effect of GnRH to lengthen LH beta mRNA, whereas 8Br-cAMP or forskolin produced the opposite effect. These data extend previous results which suggested that cAMP modulates gonadotropin secretion and indicate that the cAMP/A-kinase pathway regulates each of the gonadotropin subunit mRNAs.     

Polydipsia-induced alcohol dependency in rats: a reexamination

Seven Holtzman rats were kept on a polydipsia-induced schedule of alcohol consumption for 3 months in a replication of a 1972 study by Falk and colleagues. Contrary to their results, there was no evidence of alcoholism.     

A reexamination of the role of imagery in learning and memory.

Five experiments were conducted to examine whether the superior recall of concrete over abstract words might be better accounted for in terms of relative differences in the processing of relational and distinctive information rather than redundant verbal and imaginal memory codes. Concrete and abstract word pairs were presented in the standard paired-associated learning task or under conditions intended to affect the nature and extent of relational processing between pair members. Concreteness effects were attenuated or eliminated when relational processing was prevented at encoding (Experiments 3, 4, and 5) or when the use of encoded relations within pairs was prevented at recall (Experiments 1, 2, and 3). The results indicated the viability of an account of concreteness effects in paired-associate learning based on the joint functions of distinctive and relational information. They also remove theoretical constraints imposed on imagery theories by the incorrect assumption of a uniform presence of concreteness effects in memory for word lists. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

A reexamination of the role of nonhypnotic suggestibility in hypnotic responding.

An alcohol-placebo paradigm was used to measure individual differences in the tendency to have directly suggested, as well as unsuggested, alterations in experience. Using the spectral analytic technique of C. G. Balthazard and E. Z. Woody (see record 1992-22396-001), such alterations in experience were found to be differentially correlated with the easiest items of the Harvard Group Scale of Hypnotic Susceptibility, Form A. The results indicated that easy hypnotic performances depend on some kind of social suggestibility not unique to hypnosis, and this factor becomes less important as the difficulty of the hypnotic performances increases. The results may also shed light on hypnotic sequelae, context effects, and two-component models of hypnotic performance. (PsycINFO Database Record (c) 2011 APA, all rights reserved)     

N-methyl-D-aspartic acid stimulation of vasopressin release: role in osmotic regulation and modulation by gonadal steroids

Previous experiments demonstrated that excitatory amino acids participate in the osmotic regulation of vasopressin secretion, but the specific involvement of N-methyl-D-aspartic acid (NMDA) receptors was not evaluated. This was demonstrated in the present studies. NMDA stimulated vasopressin release from perifused explants of the hypothalamo-neurohypophyseal system (HNS), and osmotic stimulation of vasopressin release was inhibited by MK-801 (10 microM) and AP5 (100 microM) NMDA receptor antagonists. The effective concentration of NMDA was dependent upon the Mg2+ concentration of the perifusate with stimulation observed at 1 microM NMDA in Mg2+-replete compared with 5 microM in low-Mg2+ medium. Previous experiments also demonstrated that estradiol and dihydrotestosterone (DHT) inhibited osmotically stimulated vasopressin secretion, and a nongenomic mechanism of action was suggested by the ability of steroids conjugated to bovine serum albumin to replicate the effect. Experiments were performed to explore the potential role of NMDA receptors in this mechanism. Estradiol (50 pg/ml) and DHT (3 ng/ml) inhibited NMDA stimulated vasopressin release in perifused HNS explants. These results suggest a role of NMDA receptors in the mediation of vasopressin secretion in osmotically stimulated release. Furthermore, estradiol and DHT may exert their inhibitory effect on osmotically stimulated vasopressin release via the NMDA receptor.     

Autoreceptor-mediated regulation of GABA release: role of uptake inhibition and effects of novel GABAB antagonists

While the role of GABAB autoreceptors in the regulation of GABA release in synaptosomes and brain slices is well established, little is known about their role in vivo. Doubts have arisen because there is an apparent discrepancy between the frequencies at which GABA neurons fire and the frequency range within which autoreceptor regulation is observed in vitro. To see whether this apparent mismatch could be due to the use of a GABA uptake inhibitor in the release experiments in slices, we have compared the frequency dependencies of GABA release in the presence and absence of uptake inhibition. Before-hand, the previously incomplete frequency curve in the presence of uptake inhibition was extended at the lower end. To achieve this, stimulation was performed by means of groups of 4 pseudo-one-pulses (POP's) at inter-POP intervals corresponding to frequencies of 0.015625-0.5 Hz. It could be shown that activation of the GABAB autoreceptor by endogenously released GABA begins at a stimulation frequency as low as 0.0625 Hz. Experiments with the antagonist, CGP 35348, at inter-POP intervals of 1 min, at which the preceding POP has no longer an effect on GABA release during the next one, showed that basal release alone already substantially activated the autoreceptor. The frequency dependence in the absence as compared to the presence of uptake inhibition was shifted towards higher frequencies by a factor of 4. We do not consider this enough to remove our doubts about the in vivo operativity of GABAB autoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cotton fiber annexins: a potential role in the regulation of callose synthase

Cotton fibers contain a characteristic set of proteins which interact with plasma membranes in a Ca(2+)-dependent manner. The association of these proteins with the membrane is correlated with a reduced level of UDP-glucose: (1-->3)-beta-glucan (callose) synthase activity. Analysis of the proteins released from membranes by EDTA treatment shows that the most abundant proteins comprise a family of at least three polypeptides (p34) which resemble annexins. This resemblance includes similarity in size (about 34 kDa), sequence homology, Ca(2+)-dependent precipitation or interaction with the plasma membrane, and ability to serve as a substrate for phosphorylation by endogenous protein kinase(s) which also bind to the membranes in a Ca(2+)-dependent manner. A purified fraction of these annexins binds to, and inhibits, the activity of a partially purified cotton fiber callose synthase. These findings suggest that one possible function of annexin(s) in plants is to modulate the activity and/or localization of callose synthase.     

Central cardiovascular effects of tacrine in the conscious dog: a role for catecholamines and vasopressin release

Centrally acting cholinergic agents are currently reported to increase blood pressure in various species through the stimulation of muscarinic cholinoceptors. Moreover, several cardiovascular adverse effects have been reported from clinical studies. The aim of this study was to investigate the effects of tacrine, an acetylcholinesterase inhibitor which has been reported to have therapeutic potential in Alzheimer's disease, on blood pressure and two vasopressor systems (sympathetic and vasopressinergic) in Beagle dogs. Intravenous (i.v.) tacrine (2 mg kg(-1)) induced, in conscious and anesthetized dogs, an increase in systolic and diastolic blood pressure, accompanied by bradycardia. This increase was dose-dependent with a peak effect at 1.5 min following administration. Tacrine also induced an increase in noradrenaline, adrenaline and vasopressin plasma levels. Pretreatment with the muscarinic receptor antagonist, atropine (2 mg kg(-1), i.v.), abolished the pressor response to i.v. injection of tacrine while pretreatment with the peripheral muscarinic receptor antagonist, methylscopolamine (0.2 mg kg(-1), i.v.), did not alter the increase in blood pressure. Similarly, noradrenaline and adrenaline changes in plasma levels were not modified by methylscopolamine but were abolished by atropine pretreatment. A similar tendency although not significant was observed for vasopressin plasma levels. The present results demonstrate that in dogs, tacrine (2 mg kg(-1), i.v.) stimulates central muscarinic cholinoceptors to increase blood pressure through activation of the two components of the sympathetic nervous system (i.e., neuroneuronal noradrenergic and the neurohormonal adrenergic pathways) as well as through increasing noradrenaline, adrenaline and vasopressin plasma levels.     

Cytomorphological changes in the rabbit oviductal epithelium after human chorionic gonadotropin treatment

Progenitor cells were isolated from the developing human central nervous system (CNS), induced to divide using a combination of epidermal growth factor and fibroblast growth factor-2, and then transplanted into the striatum of adult rats with unilateral dopaminergic lesions. Large grafts were found at 2 weeks survival which contained many undifferentiated cells, some of which were migrating into the host striatum. However, by 20 weeks survival, only a thin strip of cells remained at the graft core while a large number of migrating astrocytes labeled with a human-specific antibody could be seen throughout the striatum. Fully differentiated graft-derived neurons, also labeled with a human-specific antibody, were seen close to the transplant site in some animals. A number of these neurons expressed tyrosine hydroxylase and were sufficient to partially ameliorate lesion-induced behavioral deficits in two animals. These results show that expanded populations of human CNS progenitor cells maintained in a proliferative state in culture can migrate and differentiate into both neurons and astrocytes following intracerebral grafting. As such these cells may have potential for development as an alternative source of tissue for neural transplantation in degenerative diseases.     

Evidence for an interaction between alpha-MSH and opioids in the regulation of gonadotropin secretion in man

BACKGROUND AND OBJECTIVES: The usefulness of testing for antibody to hepatitis B core antigen (anti-HBc) as a surrogate marker for non-A, non-B hepatitis can no longer be clearly established in the face of anti-hepatitis C virus testing. Application of anti-HBc testing in blood donors for detection of hepatitis B in addition to hepatitis B surface antigen testing (HbsAg) is a matter of debate. MATERIALS AND METHODS: We examined the serology and risk analysis data in a group of first-time blood donors. In 1.48% of 16,081 donors, anti-HBc reactivity was found. We invited a study group of 112 donors for extensive interviewing about the risk of blood transmissible diseases, and for serological testing. A control group of 240 first-time donors was studied as well. RESULTS: In the study group, the age was older (p < 0.001), a history of liver disease was more frequent (p < 0.001), and the donor (p < 0.001) or the donor's partner (p < 0.05) had either stayed longer in an HBV-endemic area or had been born in one. Combining these with the serological results, we found that strong anti-HBc reactivity was related to hepatitis B risk factors in HBsAg-negative donors. CONCLUSIONS: Anti-HBc testing in HbsAg-negative first-time donors makes it possible to identify hepatitis B risk factors with a prevalence of 0.02%. Our findings also stress the importance of including the history of the donor's partner(s) in the risk analysis before blood donation.     

Amino acid residue 250 has a functional role in the assembly of rabbit liver microsomal cytochrome P450 2B4

Cytochrome P450 2B4 lacking amino acids 2-27, CYP2B4 (delta2-27), was mutated at position 250 and expressed in E. coli fused to glutathione S-transferase. Expression of the E250S variant (holo- plus apoenzyme) proceeded to an extent comparable with that of CYP2B4 (delta2-27), while the protein level of the E250P mutant averaged 42% that of the control pigment. Comparison of these data with the corresponding reduced CO difference spectra of the various CYP2B4 (delta2-27) forms revealed that, in the control and E250S preparations, about 90% and 44%, respectively, of the total amount of hemoprotein present existed in the form of holoenzyme, whereas the E250P derivative failed to produce a reduced carbonyl complex. Thus, replacement of the negatively charged E250 with an uncharged, polar serine residue substantially hampered assembly of CYP2B4 (delta2-27); introduction of an alpha-helix-disrupting proline completely blocked the formation of holoenzyme. These phenomena suggested that the negative charge of E250, residing in the putative G helix, underwent pairing with some positively charged group, possibly H285 located in the I helix. Deletion of the negative charge obviously perturbed the active-site geometry such as to affect both the incorporation and/or retention of the heme ligand and the spectral binding of substrates such as hexobarbital.     

Ovarian control of gonadotropin hormone-releasing hormone pulse generator activity in the rhesus monkey: duration of the associated hypothalamic signal

The activity of the GnRH pulse generator in the rhesus monkey is associated with abrupt increases in multiunit electrical activity (MUA) volleys recorded from the mediobasal hypothalamus that precede each pulse of LH in the peripheral circulation. In long-term ovariectomized animals the duration of these MUA volleys is 10-25 min and consists of a brief initial 'overshoot' followed by a plateau phase that ends in a rapid decline to baseline activity. In intact monkeys, however, the MUA volley lasts only 1-3 min, a duration equivalent to the overshoot in ovariectomized animals. In addition, the maximal frequency of neuronal activity during each MUA volley is reduced in normal animals when compared to castrates. As shown in earlier studies, estradiol given to ovariectomized monkeys causes a reduction in the duration of MUA volleys to that characteristic of intact animals within 3-5 h. In contrast to this acute effect of estradiol, the increase in MUA volley duration following ovariectomy is a gradual phenomenon, 4-6 weeks being required to achieve the MUA volley duration observed in long-term ovariectomized monkeys. A similar slow time course was observed for the increase in maximal neuronal frequency during each MUA volley. This protracted effect of ovariectomy on MUA volley duration and firing rate may be the consequence of hypothalamic remodelling but this consideration must be tempered by the observation that estradiol reverses these phenomena within hours.     

Evidence for immune regulation in the induction of transplantation tolerance: a conditional but limited role for IL-4

Most experimental models of allograft tolerance depend on manipulation of immune responses at the time of transplant. In such systems, the graft itself probably plays an important role in the induction of unresponsiveness but as a consequence may suffer immune mediated damage. Ideally, recipients would be made specifically unresponsive before transplant such that the graft is protected from the outset. In this report, we demonstrate that CBA mice pretreated with donor-specific transfusion plus anti-CD4 Ab 28 days before transplant accept cardiac allografts indefinitely without further intervention. Adoptive transfer of spleen cells from mice with long term surviving grafts results in donor-specific graft acceptance in naive secondary recipients, indicating that tolerance in this system involves immuneregulation. Regulation develops as a result of the pretreatment protocol alone, since transfer of cells from pretreated but untransplanted mice to naive recipients also leads to prolonged allograft survival without additional therapy. Neutralizing IL-4 at the time of tolerance induction had no effect on graft outcome in primary recipients. However, removal of IL-4 from the adoptive transfer donors at the time of tolerance induction prevented long term engraftment in the majority of secondary recipients. Our data demonstrate that pretreatment of transplant recipients can establish immune regulation powerful enough to override the responses of an intact immune repertoire and that under stringent conditions at least, development of this regulatory population may in part be dependent on IL-4.     

Neurochemical and electrophysiological evidence for the existence of a functional gamma-hydroxybutyrate system in NCB-20 neurons

Clonal neurohybridoma NCB-20 cells express a valproate-insensitive succinic semialdehyde reductase activity that transforms succinic semialdehyde into gamma-hydroxybutyrate. This activity (1.14+/-0.16 nmol/min/mg protein) was similar to the lowest activity existing in adult rat brain. [3H]gamma-Hydroxybutyrate labels a homogeneous population of sites on NCB-20 cell membranes (Kd=250+/-44.4nM, Bmax=180+/-16.2fmol/mg protein) that apparently represents specific gamma-hydroxybutyrate binding sites characterized previously on brain cell membranes. Finally, an Na+-dependent uptake of [3H]gamma-hydroxybutyrate was expressed in NCB-20 cells with a Km of 35+21.1 microM and a Vmax of 80+/-14.2 pmol/min/mg protein. A three-day treatment with 1 mM dibutyryl-cyclic-AMP induced a three-fold increase in the cellular succinic semialdehyde reductase activity. In parallel, a K+-evoked release of [3H]gamma-hydroxybutyrate occurred. This release was Ca2+ dependent and was not present in undifferentiated cells. Cyclic-AMP treatment induced a decrease of [3H]gamma-hydroxybutyrate binding sites, which could be due to spontaneous gamma-hydroxybutyrate release. Patch-clamp experiments carried out on differentiated NCB-20 cells revealed the presence of Ca2+ conductances which were partially inhibited by 50 microM gamma-hydroxybutyrate. This gamma-hydroxybutyrate-induced effect was blocked by the gamma-hydroxybutyrate receptor antagonist NCS-382, but not by the GABA(B) antagonist CGP-55845. These results demonstrate the presence of an active gamma-hydroxybutyratergic system in NCB-20 cells which possesses the ability to release gamma-hydroxybutyrate. These cells express specific gamma-hydroxybutyrate receptors which modulate Ca2+ currents independently of GABA(B) receptors.     

Verapamil regulation of a defective SR release channel in the cardiomyopathic Syrian hamster

The Bio 14.6 Cardiomyopathic Syrian Hamster (CMH) has an autosomal recessive disease characterized by intracellular calcium overload, cardiac and skeletal myopathies and premature death from congestive heart failure. Early treatment of these animals with the calcium antagonist, verapamil (V), prevents the development of the disease. We have previously provided evidence supporting a specific defect in the ryanodine-sensitive SR calcium release channel (SRCRC) in CMH. We now provide physiologic and biochemical evidence that V modulates SRCRC. Papillary muscles prepared from F1B control hamsters (F1B) revealed an enhanced inotropic responsiveness to V and ryanodine (R) with age, not seen with CMH. CMH papillary muscles demonstrated paradoxical positive inotropic effects of V and R not shared with F1B. The positive inotropic effects of V and R were not additive. V enhanced the affinity (decreased KD) of [3H]ryanodine binding to cardiac membranes. Thus, V may prevent the overt manifestations of genetic disease in CMH by modulating a defective ryanodine-sensitive SR release channel.     

Role of catecholamines in release of gonadotrophic hormones in the rabbit

Role of catecholamines (CA) in transmission of neurohormone releasing gonadotrophin (GnHR) from the hypothalamus to hypophysis was estimated on the basis of the hypothalamic catecholaminergic system blockade with 6-hydroxydopamine (6-OHDA) and intraventricular infusions of CA in the rabbit. 6-OHDA administered intraventricularly in doses 200-500 mug caused temporary blockade of ovulation. Intraventricular infusion of noradrenaline (NA) induced ovulation in 50% of animals tested, whereas dopamine (DA) and adrenaline (A), induced ovulation only in very few cases. It seems, that catecholaminergic system participates in transmission of neurohormones to the hypophysis, and NA plays the most important role in this process.