Escherichia coli associated with acute measles and diarrhoea at Kenyatta National Hospital, Kenya.

Three hundred and three children under 5 years old with acute measles and diarrhoea (cases) and 300 other age-matched children with diarrhoea (controls) were examined for enteroadherent E. coli (EAEC) and other agents including rotavirus and Cryptosporidium. EAEC was determined by tissue culture of HEP-2 cells. Other agents were determined by conventional methods. EAEC was identified from both cases and control accounting for 10.3% (31/303) and 15.2% (46/300) respectively. Other bacterial agents were: 10.3% (31/303) from cases and 12.8% (39/300) from controls. A higher detection rate of enteroparasites was found among cases 15% (45/300) than controls 8.9% (27/300) whereas rotavirus was the reverse, 3% (9/303) in cases and 30.3% (92/300) in controls. To our knowledge characterization of EAEC has not been done before and therefore might be attributing factor to some of our unexplained diarrhoeal cases.


Nigerian secondary school teachers: a pilot survey of views and knowledge about drug abuse.

The views and knowledge level of 135 randomly selected Secondary School teachers in the Abeokuta Local Government area of Ogun State on drug abuse were sought through a self report anonymous questionnaire. They were mostly male teachers (59.3%) under the age of 30 years (89.6%). The majority (59.3%) were never exposed to drug education as students whilst less than 20% have been exposed since qualifying as teachers. 72.6% do not currently teach their students Drug education. Although the majority had a good concept of the definition of drug abuse, their view on the potential drugs of abuse and the concept of "drug dependence" were inadequate. Respondents recently exposed to drug education were more likely to identify salicylate analgesics as potential drugs of abuse at a statistically significantly level (p less than 0.05). 85.9% of the respondents chose "guidance counselling" as the approach to the management of student drug abusers despite the dearth of this professional cadre in the schools. The disquieting implications of these findings in relation to the National Campaign aimed at stemming the increasing tide of drug abuse among youths in the country were discussed and suggestions proffered on ways of rectifying these. Finally the authors gave suggestions on the pattern and content of teacher-targetted formal drug education programme in the light of the findings.


ABO blood groups distribution in relation to hepatitis B surface antigen and the presence of lipoidophil antibodies.

Three hundred and thirty donors were screened for HBs Ag using direct passive haemagglutination and the presence of Lipoidophil antibodies as detected by VDRL test. Blood group O donors had the highest HBs Ag prevalence rate of 4.3% as against the 0% frequency for group AB donors. There were no significant association between ABO blood group distribution and the presence of HBs Ag (P greater than 0.05). 11 donors in all were positive for HBs Ag giving a total prevalence of 3.3%. The sera of 27 blood donors (8.2%) contained lipoidophil antibodies. The highest percentage of VDRL test positivity was in blood group B donors (22.7%) followed by group O (9.1%). None of the 6 group AB donors had a reactive sera. Test of significance showed that there was no association between ABO distribution and the presence of lipoidophil antibodies. In conclusion, it is stated that since none of the blood donors that was reactive to the VDRL reagin carried HBs Ag, hepatitis virus B unlike other viral infection, may not elicit the production of lipoidophil antibodies.


Assessment of blood compatibility of haemodialysis membranes using a miniature flat sheet dialyser.

Available pre-clinical techniques of assessing severity of untoward reaction by blood following contact with haemodialysis membranes do not account for the effects of dialysate and flow characteristics in the observed blood changes. A miniature flat sheet dialyser that considered these effects was prepared and tested in both in-vitro and ex-vivo circuits. Changes in platelets and leucocytes in heparinised human blood in-vitro tests did not distinguish regenerated cellulose (Cuprophan 150PM) membrane from a synthetic membrane, a copolymer of acrylonitrile and sodium methyl sulphonate (AN69S). Ex-vivo tests using rats showed more marked leucocyte (41.3%) and platelet (43.1%) depletion by Cuprophan 150PM than AN69S after 90 minutes of dialysis. Leucocyte and platelet loss due to AN69S were 26.9% and 13.4% respectively. In addition, Cuprophan 150PM membranes exhibited high affinity for leucocytes and platelets in both in-vitro and ex-vivo tests compared to AN69S membranes which were primarily covered with erythrocytes. Application of simulated in-use techniques in preclinical evaluation of blood compatibility membranes that are used in extra-corporeal treatment are recommended.


Experience with carbimazole in the drug treatment of the hyperthyroidism of Graves' diseases in Nigerians.

To estimate the rate of remission of relapse in patients with thyrotoxic Graves' disease following therapy with thionamides, the course of 49 patients who were treated with carbimazole and followed personally for a mean (+/- SD) of 22.0 +/- 17.8 months, was analysed. A remission rate of 61% was obtained. Remissions were more common in patients with small goitres and those with a short duration of illness. Relapse, which was infrequent, occurred mostly within a few months following the discontinuation of the drug. 32% of the patients left the clinic either before or soon after achieving euthyroidism. It is concluded that carbimazole is quite efficacious in the induction or remission in newly diagnosed Nigerian patients with toxic Graves' disease and, at the present time, should probably be the principal treatment modality for these patients rather than subtotal thyroidectomy. Efforts should be made to reduce the frequent drop-out of our patients from the clinic during follow-up.


The response to varied timing and spacing of tetanus toxoid administration in pregnancy.

One hundred and sixty-eight pregnant women at the University of Nigeria Teaching Hospital, Enugu, Nigeria, were studied for their response to varied timing and spacing of tetanus toxoid administration. In patients who started immunization before the third trimester of pregnancy, the longer the interval between the two doses, the better the response. In contrast, when immunization was started in the third trimester, the longer the interval between the two injections, the poorer the response. In all cases, the presence of antitetanus antibodies at booking conferred better response. In addition, the titres of antibodies in all patients who responded continued to rise till delivery. The significance of these findings is discussed and recommendations made.


Meconium aspiration syndrome--unusual features.

A term infant with the meconium aspiration syndrome (MAS) is described. He had the typical clinical course of the disease despite appearing well nourished, and with good Apgar scores. At 5 months of age, he continued to do well with no sequelae. The reason for the initial passage of meconium in-utero remained speculative. The fact that the disease is preventable is emphasised.


Primary hypothyroidism with secondary hypertension: a case report.

Both primary hypothyroidism and secondary hypertension are thought to be relatively uncommon in most parts of the tropics. A Nigerian hypothyroid patient with thyroxine-responsive hypertension is reported, and this syndrome (secondary hypothyroid hypertension) is examined with respect to its possible aetiopathogenesis.


Modern concepts in the management of flail chest.

Seven patients with varying degrees of paradoxical chest wall movement (flail chest) were managed conservatively at the University of Port Harcourt Teaching Hospital with frusemide, methylprednisolone, non administration of crystalloid fluids and limitation of fluid intake. Patients showed considerable improvement within the first 24-48 hours which was sustained throughout the period of management. All seven survived and showed no signs of respiratory distress in the resting state or while performing simple exercise. One patient however had slight to moderate chestwall deformity.


Giant twin sialoliths presenting with acute cellulitis: a case report.

Sialolithiasis is a process of unknown aetiology in which calculus concretions form in the salivary gland ducts and tissue. It may occur at any age but it is most common in middle-aged adults. A case is presented of the occurrence of double giant sialoliths in the left Wharton's duct in a 16-year-old boy. The clinical symptoms and signs were somewhat atypical and included acute suppurative cellulitis in the floor of the mouth plus localized periodontitis involving 36. Under a local anaesthetic the stones were surgically extracted and healing was uneventful. Proper interpretation of symptoms and thoughtful investigation for localization of salivary stones is emphasized.


The effect of participatory school health programme on the control of malaria.

Two hundred primary level school children, aged between 7 and 18 years took part in a comparative pilot study of the effect of participatory school health education in Kisumu, Western Kenya in 1988. An experimental cohort of 100 pupils had participatory health education versus a passive control of another 100. An initial KAP survey revealed comparable sociological variables as well as morbidity statistics. No significant change occurred on the knowledge of the disease between the groups after 3 months. However, positive change in attitude here determined by positive antimalarial practices, was observed more frequently in the experimental group than in the control group. Efforts to environmental manipulation increased by 69% vs 1%, while parasite rates decreased by 32% vs an increase of 5%, respectively. Similarly there was a decrease in absenteeism of 25% vs an increase of 5% between the groups. Clinical malaria was reported less frequently in the experimental group than the control (26% against 8%). In conclusion, this pilot study suggests that participatory approach in effective health education to school children should be investigated further to determine its potential in the intersectoral malaria control strategy.


Efficacy of dapsone with pyrimethamine (Maloprim) for malaria prophylaxis in Maputo, Mozambique.

In a randomized controlled study of malaria prophylaxis, dapsone-pyrimethamine at a weekly dosage of dapsone 50-100 mg with pyrimethamine 6.25-12.5 mg or placebo was administered to 166 school children for 17 weeks. Fortnightly parasitological controls revealed 28 infections in the placebo group and none in the dapsone-pyrimethamine group. It is concluded that weekly dapsone-pyrimethamine is effective for the prophylaxis of falciparum malaria in Mozambique.


In vivo and in vitro response of Plasmodium falciparum to chloroquine in pregnant women in Kilifi district, Kenya.

In a malaria-endemic area of the Coast of Kenya with chloroquine resistant Plasmodium falciparum, we determined the prevalence and density of falciparum infection in gravid women and assessed the in vivo and in vitro parasite response to a regimen of chloroquine 25 mg/kg body weight divided over three days. P. falciparum infections were present in 65 (21.7%) of 300 pregnant women. The proportion of primigravidae who were parasitaemic was 41.8% which was significantly higher than that of the multigravidae (17.1, P less than 0.01). Primigravidae did not show a significantly higher mean parasite density than the multigravidae. The in vivo tests showed that 45.9% of all the P. falciparum infections were resistant to chloroquine predominantly at RI and RII levels with percentages 36.1% and 8.2%, respectively.


Prevalence and frequency of Giardia lamblia in children aged 0 to 60 months with and without diarrhoea.

Stool specimen results from children with diarrhoea and without diarrhoea, in a rural village community in Kenya, were analysed in order to compare prevalence and age frequency distributions of Giardia between the 2 groups. There was evidence for 2 age-related prevalence peaks for Giardia in children 60 months and below. Furthermore, there appeared to be a clear Giardia-diarrhoea relationship in the age group of 19 to 24 months.


Frequency of isolation of Cryptosporidium oocysts in Ethiopian children with acute diarrhoeal disease.

Stool specimens from one hundred children with acute diarrhoeal disease seen at the Gondar College of Medical Sciences Hospital, north-western Ethiopia were prospectively screened for the presence of oocysts of Cryptosporidium species. The study was conducted between June 1989 and January 1990. Nine out of one hundred specimens were positive for oocysts of Cryptosporidium. This study has demonstrated the presence of this parasite in the study region. A well desgned case control study is recommended to elucidate its clinical manifestations and association with malnutrition.


A preliminary study on intermediate snail hosts of schistosomes in River Enyau, Arua district, Uganda.

A total of 1260 snail intermediate hosts of both Schistosoma mansoni and Schistosoma haemotobium were collected along the non-seasonal River Enyau which lies half a kilometre west of Arua, a north-western Uganda town. The snails collected were Biomphalaria pfeifferi 709: Biomphalaria choanomphala 457, Bulinus Bulinus tropicus 94. These were screened for cercariae shedding and 25 B. pfeifferi and 12 B. choanomphalal shed mammalian cercaricae while none of the Bulinus (Bulinus) tropicus shed any cercariae. River Enyau Biomphalaria snails were susceptible to a Schistosoma mansoni strain from Entebbe. Cercariae derived from these snails and also those found infected in the River Enyau were confirmed to be S. mansoni using laboratory mice.


Arterial blood gases and acid-base status of adult patients presenting with acute severe asthma at Kenyatta National Hospital, Nairobi.

Forty adult patients with acute severe asthma were assessed for arterial blood gases and acid-base changes at presentation at the Casualty Unit, Kenyatta National Hospital, Nairobi, Kenya. Marked degrees of hypoxaemia (mean P(a)O2 of 8.02 kP(a)), hypocapnia (mean P(a)CO2 of 4.62kP(a)) with apparently normal pH (mean 7.384) were documented in the majority of these patients. Based on these findings, a significant number of the patients (68.5%) were either candidates (5.3%) or potential candidates (63.2%) for artificial ventilation. Records at this hospital suggest an increasing trend in asthma admissions and mortality. Additionally, Intensive Care Unit (ICU) admissions for asthma are associated with high mortality (54.4%). Pulsus paradoxicus emerged as the most significant (p = 0.002) clinical correlate to asthma severity. Central cyanosis was detected in none of the study patients suggesting that it is an unusual finding in the common presentation of acute severe asthma at this hospital. Its clinical detection would therefore imply an ominous clinical state. It is recommended that clinicians should take every opportunity to adequately assess their asthma patients before prescribing medications. They are also encouraged to more frequently request for blood gas studies in acute asthma. Those in charge of clinical teaching should direct appropriate efforts towards improved understanding of the pathophysiology and interpretation of acid-base disorders.


Kagera 1989 health survey: 1. Human immunodeficiency virus seroprevalence in adolescents.

One thousand four hundred and fifty five adolescents were surveyed for Human Immunodeficiency Virus (HIV) infection in an area experiencing AIDS epidemic. The study was done in order to estimate control measures. Among the 1455 adolescents serosurveyed, 47 (3.2%) were HIV infected, of whom 30 (63.8%) were all aged 10-15 years old. The male/female ratio was 1:1. Of all the adolescents examined 830 (57.1%) were attending school. Among the selected potential risk factors namely non-school enrollment, illicit injections, blood transfusion and sexually transmitted diseases none of these was significantly associated with HIV seropositivity. Special care should be taken when interviewing adolescents on sensitive issues like the ones associated with HIV infection. In this study potential risk factors for getting HIV infection were rarely reported, thus making it difficult to get significant association with the HIV infection.


In vitro experimental antileishmanial potential of ethylenediamine tetraacetic acid, disodium salt.

Leishmania donovani promastigotes (NLB 065) were treated to various concentrations of ethylenediamine tetraacetic acid (EDTA), disodium salt for 7 days. Concentrations of up to 1.0 mg/ml produced no significant reduction in the population of Leishmania promastigotes cultured in RPMI 1640 medium supplemented with 15% foetal calf serum and incubated at 27 degrees C (P greater than 0.05). Concentrations between 0.05 and 0.1 mg/ml EDTA did not significantly affect the viability of Balb/c mouse peritoneal macrophages (P greater than 0.05) whereas those equal to or above 0.2 mg/ml were severely toxic to macrophages (P less than 0.05; n = 8). Leishmania parasites engulfed in mouse peritoneal macrophages were treated to various concentrations of ethylenediamine tetraacetic acid (EDTA) disodium salt. The higher the EDTA concentration within the acceptable toxic levels for macrophages (less than or equal to 0.1 mg/ml), the higher was the rate at which parasites were cleared (p less than 0.05). The length of time in days within which infected macrophages were treated contributed significantly to a decline in the level of parasite-loads (P less than 0.05; n = 10). Analysis of mean metal ion values in mg/1 using atomic absorption spectrometry revealed that the levels of calcium, magnesium, and iron were virtually unchanged in the supernatant of variant culture experiments (P greater than 0.05). The level of manganese was higher in ethylenediaiene tetraacetic acid (EDTA), disodium salt-treated cultures as compared to the control (P less than 0.05; n = 20).(ABSTRACT TRUNCATED AT 250 WORDS)


Thyroid hormone profiles in patients with chronic renal failure on conservative management and regular haemodialysis.

Patients with chronic non-thyroid illness (NTI) generally show biochemical features of hypothyroidism despite their being clinically euthyroid. Using assays that employ the antibody coated tubes technique, we measured thyroid hormone levels in 52 patients with chronic renal failure. 30 of the patients were on conservative management while 22 were on regular haemodialysis (HD) for a mean period of 14.2 +/- 9.9 months. The findings were compared to those of 14 healthy controls. All patients were clinically euthyroid. Only 3 patients on HD had goitres. The mean value of total thyroxine (TT4) total triiodo thyronine (TT3), free thyroxine (FT4) and the free triiodothyronine (FT3) were low in patients compared with controls (P less than 0.01). The mean basal thyroid stimulating hormone (TSH) levels were significantly higher in patients than in controls (P less than 0.001). There were no significant differences in all parameters between the patients on conservative management and those on haemodialysis (P greater than 0.05). Although all patients were clinically euthyroid, the biochemical features suggest hypothyroidism.


Oral health habits and periodontal health among a group of university students in Kenya.

The purpose of this study was to determine the oral health habits and periodontal health among a group of university students in Kenya. The study comprised 243 randomly selected first year students, mean age 23 years, at the University of Nairobi. Questionnaire response showed that 39% of the participants had visited a dentist before, mainly for extraction. Sixteen per cent said they visited a dentist at least once a year and 96% said they brushed daily. Clinical examination was done in a room with natural daylight using the WHO criteria. Plaque was visible on 35% of the surfaces. 11% of gingival sites examined bled on probing. Only one participant had pocket depth greater than or equal to 4 mm. Supra- and sub-gingival calculus was recorded at 18% of index tooth surfaces. The study will be considered as a baseline for later studies on oral health trends in similar populations.


Rehabilitation of the blind: a review.

Rehabilitation of the blind is an active process whereby the blind person is helped to become an independent and useful member of the community. Rehabilitation thus improves the psychological wellbeing of the blind person as well as making him a happy, contributing member of the society.


Multiple sexually acquired diseases occurring concurrently in an HIV positive man: case report, diagnosis and management.

A case of an HIV positive man with multiple sexually acquired disease occurring concurrently is described. Risk behaviours that could have predisposed him to HIV infection are discussed. The factors which might have interacted to make the sexually acquired infections severe and difficult to treat are postulated.


[A comparison of EEG mapping with and without visual artefact control in focal cerebral lesions].

In 163 patients with focal cerebral lesions, 43 of them with completed stroke, 43 patients with TIA, 33 patients with ICH and 29 patients with malignant and 15 patients with benign tumors EEG mapping and CT was performed. The results of EEG mapping obtained using automated artifact detection were compared to those achieved by means of visual control of raw EEG. Furthermore the impact of long (850 +/- 250s) or short (32s) analysis time was studied. Eliminating artifacts by means of visual control of raw EEG significantly more positive results were obtained than using automated artifact detection. That was found in patients with as well as in patients without lesions in CT. In relation to etiology a significant difference was found only in cerebrovascular diseases but not in the other patients-groups. Corresponding results were found in 75% of the patients. The visual control provided additional lateralization especially in patients with CS (37%) and TIA (26%). A longer duration of analysed EEG epochs did not increase the number of focal changes in EEG mapping.


[The value of EEG mapping in intracerebral hematomas].

33 patients with intracerebral hemorrhage were studied and the findings of EEG mapping, routine EEG and CT were compared. It could be shown, that the EEG mapping revealed in 6 patients additional focal changes corresponding with the clinical signs compared to conventional EEG. Identical results of EEG mapping and CT were found in 27 patients. In 5 patients the focal changes were localised contralateral to the hyperdense lesion in CT, and in 1 patient the EEG mapping failed to show any lateralisation. A negative intercorrelation between the size of the hematoma and the frequency, which revealed the focal signs in EEG mapping, could be shown.


[The dependence of rapid voluntary contractions on the tremor phase in parkinsonism].

Parkinsonian patients sometimes have problems to produce really fast motor responses. Often, these are significantly delayed in comparison to those of normal controls. In patients with tremor-at-rest, this delay might be induced by some attraction of the movement execution to the tremor oscillator, according to a hypothesis of Hallet et al. (1977). This study now examined the phase relationship between the on-going tremor and the onset of fast voluntary contractions in Parkinson's disease (PD), quantitatively. Five patients with prominent tremor-at-rest performed rapid isometric index finger abductions under self paced (SP) and reaction time (RT) conditions, and force and surface EMG signals from the FDI muscle were analyzed. In both conditions the SP and the RT, data showed that the onset of the contraction mostly occurs during the descending phase of the tremor oscillation and, accordingly, the phasic EMG burst synchronously with the tremor bursts confirming the above mentioned hypothesis.


[Event-related negativity and alpha band desynchronization in motor reactions].

The foreperiod of a motor reaction to an impending stimulus is accompanied by a slow negative wave of the event-related potential, the "contingent negative variation" (CNV), when the imperative stimulus follows at a fixed interval to a warning stimulus. Using similar experimental paradigms, an event-related desynchronization (ERD) in the alpha range occurs indicating cortical activation before and during motor acts. Both EEG-parameters were quantified for four subjects using the following experimental design: One second after a non-informative warning signal (WS) a cue was presented that indicated by which hand the subject had to respond quickly after presentation of a reaction stimulus (RS); subjects were requested to respond by pressing a button with their left or right thumb. EEG-signals were recorded unipolarly from 17 electrodes. Average ERPs and ERD were computed for all electrode sites for trials with left and right hand movements, respectively, and topographically displayed in simultaneous amplitude and ERD-maps. CNV and ERD could be observed simultaneously over similar brain regions, namely central over the motor cortex, frontal with possible location over the supplementary motor area (SMA) and also covering the parietal region. While the CNV had a interindividually different and wide distribution, the ERD calculated for the upper alpha band mapped focally over the electrode sites C3 and C4 overlaying primary sensorimotor structures, with a contralateral dominance. In contrast to the negativity which is terminated following delivery of the response stimulus, the ERD persisted during the motor reaction showing a similar pattern some hundreds of milliseconds before and after movement onset.(ABSTRACT TRUNCATED AT 250 WORDS)


[Are cognitive processes in a phase relation with 40 Hz EEG?].

A coherent EEG-rhythm occurring in the 40 Hz range has been shown to correlate with cognitive processes of attention in a large number of studies. We intended to evaluate whether this frequency is of specific functional significance, and whether it may represent a rhythm of excitability for cognitive processes. The model of a rhythm of excitability assumes the 40 Hz-EEG to be a timer and organizer for information processing. Event-related potentials of 12 healthy normal subjects were recorded in an experiment of attention (auditory click stimulation). In order to evaluate whether the phases of the 40 Hz-EEG represent a rhythm of excitability, responses to stimuli having similar phases at the beginning of the stimulus were selected and averaged. An effect of the different 40 Hz-phases could not be demonstrated on the amplitude and latency of N100 and P200, components of the event-related potential associated with information processing. These findings suggest that a possible timer for cognitive processes of attention is not linked to the phases of the 40 Hz-surface-EEG.


[P300 and dementia: diagnostic relevance II].

Recent research on the relationship between ERP-latencies and reduced information process capabilities with demented subjects has yielded divergent results. In the present study P300-latencies and RT are used in an attempt to differentiate between demented and nondemented subjects. Based on the "feature-integration-theory" of Treisman an experiment was designed in order to investigate more complex cognitive processes. The results showed that both P300-latency and RT may be used as additional diagnostic indices for cognitive capability.


[The prognostic place of EEG in comparison with evoked potentials in severe hypoxic brain damage].

This retrospective study comprises 34 patients, who died due to the cerebral damage caused by severe anoxia, who were assessed by EEG and AEP or SEP on the same day and in whom at least one of these electrophysiological methods indicated a fatal prognosis. EEG-findings identified 76% of patients with fatal outcome, whereas SEP reliably predicted the outcome in 93% of patients, who died of the cerebral anoxia. In some patients centrally acting drugs made EEG-based but not SEP-based prognosis impossible. In some cases of fatal brain damage, however, only the EEG-findings as opposed to SEP-findings predicted the fatal outcome. Thus the combined assessment by EEG and SEP appears to be optimal in the assessment of an early prognosis. The BAEP were of less prognostic value than the EEG. BAEP findings indicating a fatal outcome were to be found mainly in final clinical stages with loss of brainstem function, whereas EEG as well as SEP identified a considerable number of fatal cases in spite of comparably favourable clinical findings. The evaluation of early electrophysiological testing done within the first 3 days after the anoxic event results in an identical estimation of the prognostic significance of EEG versus BAEP- or SEP-findings, respectively.


[Acoustic evoked brainstem potentials--patterns of stimulus artefacts in irreversible coma].

Brainstem auditory evoked potentials (BAEP) were performed in a total of 20 subjects (mean age 33.2 +/- 15.1 years; severe head injury in most cases) with the diagnosis of coma dépassé. The control group consisted of 33 healthy volunteers (mean age 26.9 +/- 5.3 years). The presence, latency and amplitude of the single BAEP-components and the variations of the stimulus artifact were evaluated. The mean amplitudes of the stimulus artifact of the brain dead subjects were calculated (means +/- SD) to be 0.26 +/- 0.12 microV (control group: 0.11 +/- 0.03 microV). Pathological alterations of the mechanics of the inner ear as well as extracerebral changes in conductivity or stimulus related depolarization processes in extracerebral parts of the auditory system were taken into consideration to be possible reasons for this significant (p less than 0.001; t-test) difference.


[Do encephalo-electric signals determine the pulse frequency in man?].

One of the most interesting problems in EEG analysis is the question of the genuine generator of these biosignals. Each spontaneous EEG sampled by surface electrodes looks like a summary of stochastically distributed encephalo-electrical events. But analysing these data depending of the time distance of ECG R-waves we found signals which contain the information about the occurrence of the next ECG R-wave. The results make dubious the discussion about the autonomic character of sinus excitation as the heart pacemaker.


[Spatial distribution of the action potentials of the sural nerve].

We investigated the spatial distribution of the sural nerve sensory nerve action potential (SNAP) in 25 healthy subjects between 21 and 50 years. Stimulation was achieved through surface electrodes at the lateral malleolus. Recordings were made 15-18 cm proximal to the site of the stimulation from different positions on a line perpendicular to the sural nerve using needle electrodes insulated except for the tip. The amplitude of the SNAP decreased on both sides of the potential of highest amplitude. 10 mm lateral to the potential of highest amplitude the mean amplitude was reduced to 55%. The latency of the first positive phase decreased within increasing distance from the largest SNAP. We explained this with different influence of more distal, earlier depolarized nerve segments on the locally generated SNAP. In 20 subjects we performed an additional recording using an uninsulated needle electrode that was placed 3-4 cm subcutaneously perpendicular to the sural nerve. The latency of the SNAP recorded in this way was similar to the latency of the largest SNAP using the insulated needle electrode; however, the amplitude was smaller by 19%. We recommend for clinical practice to use the uninsulated needle in case the SNAP is smaller than 4 microV.


[The tibial evoked potential in healthy children and adolescents: normal values].

The somatosensory evoked cortical potentials were investigated for 35 healthy children (17 boys, 18 girls) between 6 and 17. The stimulation of the N. tibialis was affected at the Malleolus medialis using surface electrodes; the derivation was likewise conducted according to the ten-twenty system using surface electrodes. Investigations carried out employing this methodology demonstrate that the SEP's of the N. tibialis from the N30 to the N70 are clearly measurable in the children. The N30, P40 and N50 could be presented with the smallest standard deviations and side differences. The latencies of N30 and P40 show significant dependencies on the height of the children. Influence of gender could be demonstrated more for the latency of the N30 in from of longer latencies in boys that in girls which were independent of body size.


Effects of activin and follicle-stimulating hormone (FSH)-suppressing protein/follistatin on FSH receptors and differentiation of cultured rat granulosa cells.

The aim of this study was to investigate the actions of both activin and FSH-suppressing protein (FSP)/follistatin either alone or in combination on FSH receptor number and on the responsiveness of granulosa cells to FSH and LH. Granulosa cells were harvested from diethylstilbestrol-treated immature Sprague-Dawley rats and cultured 48 h in serum-free medium with or without treatment. Activin treatment alone (3-100 ng/ml) resulted in a 4-fold increase in FSH receptor number with no change in binding affinity. This effect of activin was inhibited 31% by FSP (100 ng/ml) treatment which alone had no effect on FSH receptor number. Treatment with activin (100 ng/ml) prevented FSH-induced down-regulation of FSH receptor number, whereas at lower concentrations (3-30 ng/ml) activin enhanced down-regulation of FSH receptor number by 20% (P less than 0.05). In contrast, FSP alone prevented FSH-induced down-regulation by increasing FSH receptor number up to 40-50%. Pretreatment of granulosa cells with activin, but not FSP, for 24 h increased the responsiveness of cells to FSH (20 ng/ml) and LH (40 ng/ml) shown by increases in aromatase activity, progesterone, and immunoreactive inhibin production over and above control in a manner which depended upon activin doses. We conclude that 1) activin enhancement of FSH action on rat granulosa cells may be mediated in part via regulation of FSH receptor number, and 2) the effects of FSP on granulosa cells are likely to be due to its activin binding properties.


Secretion and hormonal regulation of interleukin-6 production by mouse uterine stromal and polarized epithelial cells cultured in vitro.

Uterine stromal (USC) and uterine epithelial (UEC) cells were isolated from immature and mature mice to determine their ability to secrete interleukin-6 (IL-6) in response to ovarian steroids, IL-1 alpha, and soluble products produced by the heterologous cell type. In addition, the effect of IL-6 on embryo attachment and outgrowth in vitro was determined. UEC cultured on nitrocellulose filter inserts in a polarized manner secreted IL-6 with a 2.5- to 5-fold apical vs. basal preference, as determined by a B9 hybridoma cell proliferation assay and enzyme-linked immunosorbent assay. The hormonal status of animals at the time uteri were removed did not influence subsequent secretion of IL-6, as UEC isolated from immature, diestrous, and estrous stage mice exhibited both a similar amount and had a similar apical preference for secretion of IL-6. The addition of 17 beta-estradiol (E) to UEC cultures markedly inhibited total IL-6 secretion, but did not affect vectorial secretion. The inhibitory effect of E on IL-6 secretion by UEC was consistent with an apparent decrease in IL-6 transcript observed by a reverse transcriptase polymerase chain reaction assay. Other transcripts detected by this assay in UEC included IL-1 alpha, but not IL-1 beta or tumor necrosis factor-alpha. Secretion of IL-6 by UEC was not stimulated by IL-1 alpha, conditioned medium from USC, or coculture with USC. USC secreted IL-6, and while this also was inhibited by E, progesterone was more effective in this regard at physiological concentrations. In addition, there was a synergistic effect of E plus progesterone on inhibition of IL-6 secretion by USC. Secretion of IL-6 by USC was stimulated by IL-1 alpha, and coculture studies demonstrated the ability of UEC to stimulate a several-fold increase in IL-6 secretion by USC. The cytokine transcripts detected in USC cultures included IL-6 and IL-1 alpha, but not IL-1 beta. Transcripts for tumor necrosis factor-alpha were present in USC only after culture with IL-1 alpha. IL-6 added to blastocysts on laminin-coated tissue culture wells resulted in a transient inhibition of the rate of blastocyst attachment and, to a greater extent, an inhibition of the rate of embryo outgrowth. In addition, IL-6 inhibited the size of embryo outgrowths at 24 and 48 h of culture.(ABSTRACT TRUNCATED AT 400 WORDS)


Central corticotropin-releasing hormone activates the sympathetic nervous system and reduces immune function: increased responsivity of the aged rat.

CRH acts within the brain to activate the sympathetic nervous system and reduce cellular immune function. To determine the effects of age on CRH-induced elevations of sympathetic activity and suppression of immunity, we examined the responses of plasma catecholamines, neuropeptide-Y (NPY), corticosterone, and splenic natural killer (NK) activity after microinjection of rat CRH (200 pmol) into the lateral ventricle of aged (24-month-old) Fischer 344 (F344) rats compared to those in young (4-month-old) F344 rats. Basal concentrations of plasma norepinephrine and NPY were higher in the aged than in the young animals. In addition, CRH produced a greater elevation of plasma levels of catecholamines and NPY, which persisted for a longer period of time in the aged rats compared to responses in the young animals. Splenic NK activity showed an age-related decrement at baseline, and CRH induced a further significant (P less than 0.01) reduction of lytic activity in the aged rats, but did not alter cytotoxicity in the young rats. Corticosterone basal levels and responses were similar in the aged and young rats. These results show an age-related increase in autonomic outflow and suppression of NK activity after central CRH administration. In aged animals, the central nervous system may have a role in abnormal regulation of sympathetic activity and suppression of natural cytotoxicity in vivo.


Carboxypeptidase H processing and secretion in rat clonal beta-cell lines.

Carboxypeptidase H (CP-H, EC 3.4.17.10) removes C-terminal basic amino acids from insulin and many other peptide hormone intermediates in endocrine and neural tissues. CP-H was studied in two rat insulinoma-derived clonal beta-cell lines that were chosen in part because their profile of hormone production is restricted. Although RIN 1046-38 cells predominantly produce insulin and RIN 1046-44 cells contain no known polypeptide hormone, both cell lines contain comparable levels of CP-H. The distribution of CP-H in RIN cells differs from primary tissues since only 25% of cellular CP-H is soluble whereas approximately 75% is associated with the membrane fraction. Immunoblotting with both N- and C-terminal antibodies detects a single 54 kilodalton (kDa) band corresponding to the mature form of CP-H in soluble and membrane RIN cell extracts and incubation media. Pulse-chase analysis shows that a 56 kDa precursor is initially synthesized and rapidly processed to a mature 54 kDa form. Newly synthesized, mature CP-H is detectable in the media by 30 min. Approximately 50% is retained within cells after 3 h of incubation. Secretion of CP-H activity from both RIN cell types and insulin from 1046-38 cells is acutely stimulated by membrane depolarization with KCl and attenuated by 10 mM MgCl2. Dense core secretory vesicles characteristic of the regulated secretory pathway are evident on transmission electron microscopy of both RIN cell lines. Thus, mature CP-H exists primarily as a membrane-associated form in RIN cells, but all forms of CP-H, including soluble and secreted enzyme, retain an intact C-terminus.


Insulin-like growth factor-I supports formation and activation of osteoclasts.

Although the action of insulin-like growth factor-I (IGF-I) on bone formation has been extensively investigated, the effect of the factor on bone resorption is little known. We first examined the effect of IGF-I on bone resorption by preexistent osteoclasts by using unfractionated bone cells cultured on dentin slices. IGF-I had a dose-related effect of stimulating bone resorption by preexistent osteoclasts, whereas IGF-II did not. When IGF-I was added to cultures of bone cells after preexistent osteoclasts had degenerated on the dentin slices, IGF-I increased the number of osteoclastic multinucleate cells (MNCs) with tartrate-resistant acid phosphatase activity. Moreover, IGF-I augmented the area of pits produced by newly formed osteoclasts. These results suggest that IGF-I directly or indirectly stimulates osteoclast recruitment and activation. Therefore, we next examined the direct effect of IGF-I on osteoclastic MNC formation by using hemopoietic blast cells. In the presence of 1,25-dihydroxyvitamin D3, IGF-I, like granulocyte-macrophage colony-stimulating factor (GM-CSF), dose-dependently increased the number of TRAP-positive MNCs. This stimulatory effect of IGF-I was additive with that of GM-CSF. Both IGF-I and GM-CSF supported the survival of the blast cells, indicating that IGF-I as well as GM-CSF are supporting factors for osteoclast differentiation. In addition, the blast cells possessed high affinity binding sites for IGF-I, with a Kd of 0.8 nM. These data, thus, indicate that IGF-I stimulates osteoclastic bone resorption through its direct or indirect action of supporting the generation and activation of osteoclasts.


Effect of vasotocin on cytosolic free calcium concentrations in frog adrenocortical cells in primary culture.

In a previous report we have shown that vasotocin (AVT), the amphibian counterpart of vasopressin, is a potent stimulator of corticosterone and aldosterone secretion by frog adrenocortical cells. We have also observed that the stimulatory effect of AVT on corticosteroid secretion is mediated through activation of receptors positively coupled to phospholipase-C. In the present study we examined the effect of AVT on cytosolic Ca2+ concentrations ([Ca2+]i). Since the interrenal (adrenal) gland of the frog is composed of a mixed population of chromaffin and adrenocortical cells, cytochemical identification of cultured cells was performed by immunofluorescence, using antibodies to AVT or 11 beta-hydroxylase as markers of chromaffin cells or steroid-producing cells, respectively. Cultured interrenal cells were loaded with the fluorescent Ca2+ indicator indo-1, and variations in [Ca2+]i were studied using dual emission wavelength microfluorimetry. Exposure of adrenocortical cells to AVT induced elevation of [Ca2+]i. Prolonged infusion of AVT caused an immediate increase in [Ca2+]i, followed by a sustained response of adrenocortical cells. Repeated pulses of AVT resulted in a gradual decline in the [Ca2+]i increase, suggesting the existence of a desensitization phenomenon. The effect of AVT on calcium mobilization was totally blocked when the cells were incubated in the presence of the V2 antagonist [d(CH2)5,D-Phe2,Ile4,Ala9-NH2]AVP. In calcium-free medium, the AVT-evoked increase in [Ca2+]i was suppressed. In contrast, when Ca2+ was replaced by Mn2+ in the incubation medium, the early response of the cells (transient peak of [Ca2+]i) was preserved, while the plateau phase disappeared. Incubation of the cells with the dihydropyridine Ca2+ channel blocker nifedipine did not affect the AVT-induced [Ca2+]i rise. These results indicate that AVT exerts a dual action on [Ca2+]i in frog adrenocortical cells. The initial rise of [Ca2+]i can be ascribed to immediate mobilization of intracellular Ca2+ stores, probably mediated by inositol trisphosphategated channels, whereas the sustained increase in [Ca2+]i results from nifedipine-insensitive plasma membrane Ca2+ channels.


Superinduction of c-fos gene expression by estrogen in cultured guinea-pig endometrial cells requires priming by a cycloheximide-dependent mechanism.

The c-fos gene expression is rapidly induced by various mitogenic agents and protein synthesis inhibitors in many cell types. Estradiol-17 beta can induce c-fos gene expression in breast cancer cell lines and in the uterus in vivo, but not in cultured guinea-pig endometrial cells. Using this model, we investigated whether a protein synthesis inhibitor, cycloheximide, could induce the c-fos gene and permit a superinduction by estrogens. In the presence of cycloheximide (10 micrograms/ml), protein synthesis was inhibited at 95% within the first hour. From 190 min after the addition of estradiol-17 beta or diethylstilbestrol (10(-8) M) and cycloheximide (10 micrograms/ml), there was a significant increase (ranging from 3- to 5-fold) of the c-fos messenger RNA level (2.2 kilobase in size), compared with the level in cells treated with cycloheximide alone. Nonestrogenic steroid hormones and estradiol-17 alpha were unable to induce c-fos gene expression in the presence of cycloheximide. The effect of estradiol-17 beta observed in the presence of cycloheximide was completely abolished by 4-hydroxy-tamoxifen or by Ly 156758 or by ICI 164384 (10(-6) M). The c-fos mRNAs were rather stable in cells treated with cycloheximide for 2 h (half-life = 51 +/- 6 min) and there was no further increase in the c-fos messenger RNA stability after the addition of cycloheximide plus estradiol-17 beta (half-life = 40 +/- 3 min). The overall results suggest a response at the transcriptional level. In conclusion, cycloheximide transmits activating signals to the c-fos gene which act as priming elements to allow the estrogen action in cultured guinea-pig endometrial cells.


Developmental changes in levels of luteinizing hormone receptor and androgen receptor in rat Leydig cells.

To further assess the hormonal response capabilities of Leydig cell progenitors (PLC) from 21-day-old rats, their levels of LH and androgen receptors (LH-R and AR) were measured and compared to those of isolated immature (ILC) and adult Leydig cells (ALC) from 35- and 90-day-old rats, respectively. Levels of LH receptor were estimated by Scatchard analysis of binding to [125I]hCG, and levels of LH receptor mRNA were determined by Northern blot analysis using a rat LH receptor antisense RNA probe. The numbers of LH receptors per cell measured by the binding study were 2,623 +/- 1,110 in PLC, 9,024 +/- 1,992 in ILC, and 39,896 +/- 15,234 in ALC (mean +/- SEM of four replicate experiments; ALC significantly greater than either PLC or ILC at P less than 0.05). The Northern blotting revealed three major bands [6.7, 2.6, and 2.3 kilobases (kb)] that were present in Leydig cells at all three ages and were not detected in HepG2 cells. When the steady state levels of the predominant 6.7-kb species were normalized to actin mRNA, PLC were 6.3-fold lower than ILC and 1.7-fold lower than ALC (n = 3 replicate isolations of poly(A) RNA). The 2.6- and 2.3-kb species exhibited similar trends. Levels of AR were estimated by immunoblotting using a polyclonal antibody against a synthetic peptide of the receptor (residues 14-32) that detected a 110-kilodalton AR protein. Levels of AR mRNA were estimated by Northern blot analysis, using a rat AR antisense RNA probe that detected a single 10-kb AR mRNA. The relative levels of AR protein were 1.0, 1.5, and 0.5 in PLC, ILC, and ALC, respectively (n = 3). Similar trends were observed for AR mRNA (n = 3). The observation that both LH and AR levels were lower in PLC compared to ILC is consistent with the hypothesis that the former are progenitors of Leydig cells.


Characterization of 3T3-L1 storage lipid metabolism: effect of somatotropin and insulin on specific pathways.

TLC of total cellular lipids showed that 3T3-L1 cells predominantly accumulate triglycerides. GH and insulin principally regulated glucose utilization for synthesis of the fatty acid portion of 3T3-L1 triglycerides, with little effect on glucose utilization for synthesis of other lipids or triglyceride glycerol. Gas chromatographic mass spectrometry (MS) showed that 50% of 3T3-L1 triglyceride fatty acids are 16-carbon chains. However, 3T3-L1 adipocytes were unusual, in that 20% of their triglyceride fatty acids were C15 or C17 in length, observed by both MS of saponified triglycerides and electrospray MS of intact triglycerides. Gas chromatographic analysis of 14C-labeled fatty acids showed 3T3-L1 utilization of [14C]glucose for synthesis of C15 and C16 fatty acids. Insulin and GH regulated the amount of [14C]glucose incorporation into C15 and C16 fatty acids without altering their relative ratio. GH antagonized all studied insulin-regulated events. GH antagonism of insulin-stimulated 3T3-L1 glucose transport, glucose oxidation, and glucose utilization for lipid synthesis reached a plateau of 50-60% inhibition at 0.1-0.2 nM. Insulin, at 0.1 nM, suppressed 3T3-L1 generation of glycerol from lipolysis by approximately 50%. GH, between 0.04-1.0 nM, fully reversed the insulin-inhibited lipolysis, although GH did not stimulate lipolysis beyond that in untreated control cultures. The results suggest that GH regulates a very early event in the insulin signal transduction pathway, such that is affects all insulin-responsive processes to essentially the same extent.


Requirements of follicle structure for thyroid hormone synthesis; cytoskeletons and iodine metabolism in polarized monolayer cells on collagen gel and in double layered, follicle-forming cells.

Thyroid cells take up iodine and synthesize thyroid hormones. Thyroid cell polarity plays an important role in the uptake of iodine. However, we do not know whether polarity itself is enough for thyroid hormone synthesis or whether follicle structure is required for it. Using polarized monolayer porcine thyroid cells, cultured on collagen-coated filters, and double layered, follicle-forming cells, we analyzed the relationships of iodine metabolism and cell polarity (and follicle formation). We demonstrated that follicle structure was required for thyroid hormone synthesis. A quick-freezing and deep-etching method revealed the three-dimensional ultrastructures of cytoskeletons in the thyroid cells. On the collagen gel, the thyroid cells are reorganized into polarized monolayer cells; the basal cell membranes are in contact with the collagen gel and the apical ones face the culture medium. Actin microfilaments predominate under the apical cell membranes and intermediate filaments in the basal cytoplasm. The arrangement of these cytoskeletons determines the polarity of the cells. When the cells are cultured as double layers, follicle structures are reconstructed between the two monolayers. When apical cell membranes are in contact with other apical ones or when the cells are cultured as double layers, the cells are reorganized into follicles; the basal cell membranes are in contact with the collagen gel, and the apical ones face the follicle cavities. Actin microfilaments predominate at the apical cell membranes and intermediate filaments in the basal cytoplasm. Polarized thyroid cells transport iodine from the basal compartments to the apical ones, but cannot organify iodine into thyroid hormones. However, follicle-forming cells, cultured as double layers, take up iodine and organify it into thyroid hormones. Polarity is important for iodine uptake, and follicle structure is required for thyroid hormone synthesis.


Parathyroid hormone (PTH)-related protein, PTH, and 1,25-dihydroxyvitamin D in dogs with cancer-associated hypercalcemia.

Circulating N-terminal PTH-related protein (PTHrP), N-terminal PTH, and 1,25-dihydroxyvitamin D [1,25-(OH)2D] concentrations were measured in normal dogs and dogs with cancer-associated hypercalcemia (CAH), parathyroid adenomas, and miscellaneous tumors. PTHrP was undetectable (less than 1.8 pM) in normal dogs and increased in dogs with CAH due to adenocarcinomas derived from apocrine glands of the anal sac (44.9 +/- 27 pM), lymphoma (8.3 +/- 4.4 pM), and miscellaneous carcinomas (13.3 +/- 11.4 pM). The PTHrP concentration decreased in dogs with lymphoma and anal sac adenocarcinomas after successful treatment of CAH. The PTHrP concentration had a significant linear correlation with total serum calcium in dogs with anal sac adenocarcinomas and hypercalcemia, but not in dogs with lymphoma and hypercalcemia. Serum N-terminal PTH concentrations were usually in the normal range (12-34 pg/ml) for all groups of dogs except dogs with parathyroid adenomas (83 +/- 38 pg/ml). The serum PTH concentration increased after successful treatment of CAH. Serum 1,25-(OH)2D concentrations were decreased, normal, or increased in dogs with CAH, and 1,25-(OH)2D levels decreased after treatment of CAH. In summary, circulating concentrations of PTHrP are consistently increased in dogs with CAH, and PTHrP appears to play an important role in the induction of hypercalcemia.


Cellular mechanism of metformin action involves glucose transporter translocation from an intracellular pool to the plasma membrane in L6 muscle cells.

The effects of the oral hypoglycemic drug metformin on glucose and amino acid transporter activity and subcellular localization of GLUT1 and GLUT4 glucose transporters were tested in cultured L6 myotubes. In muscle cells preexposed to maximal doses of metformin (2 mM, for 16 h), 2-deoxyglucose uptake was stimulated by over 2-fold from 5.9 +/- 0.3 to 13.3 +/- 0.5 pmol/min.mg protein. Uptake of the nonmetabolizable amino acid analog methylaminoisobutyrate was unaffected by treatment with the drug under identical conditions. Extracellular calcium was required to preserve the full response to the biguanide. Exposure of muscle cells to insulin in the presence of metformin resulted in further activation of 2-deoxyglucose transport. The latter effect was additive to the maximum effect of metformin, suggesting that the biguanide stimulates hexose uptake into muscle cells by an insulin-independent mechanism. Glucose transporter number quantified by performing studies of D-glucose-protectable binding of cytochalasin-B in plasma membranes (PM) and internal membranes (IM) prepared from L6 myotubes revealed that a 16-h treatment with 800 microM metformin significantly elevated glucose transporter number in the PM (by 47%), with an equivalent decrement in glucose transporter number (47%) in the IM. Western blot analysis using antisera reactive with the GLUT1 and GLUT4 isoforms of glucose transporters showed that metformin caused a reduction in GLUT1 content in the IM fraction and a concomitant increase in the PM. Unlike insulin, metformin treatment had no effect on the subcellular distribution of GLUT4. We propose that the molecular basis of metformin action in skeletal muscle involves the subcellular redistribution of GLUT1 proteins from an intracellular compartment to the plasma membrane. Such a recruitment process may form an integral part of the mechanism by which the drug stimulates glucose uptake (and utilization) in skeletal muscle and facilitates lowering of blood glucose in the management of type II diabetes.


Mechanism of angiotensin II-induced proliferation in bovine adrenocortical cells.

The peptide hormone angiotensin-II (AII) is a potent vasoconstrictor and major regulator of aldosterone synthesis. In addition, AII also has growth-promoting effects. We have recently shown that the lipoxygenase (LO) pathway of arachidonic acid plays a major role in AII-induced aldosterone synthesis in adrenal glomerulosa cells. The LO pathway is also involved in the vasopressor and renin-inhibitory effects of AII. However, the role of LO products in AII-induced mitogenic effects have not yet been investigated. In the present studies we have evaluated the role of the LO pathway in AII-induced proliferative responses in a bovine adrenocortical cell clone termed AC1 cells. In addition, the potential receptor type and mechanism of AII-induced proliferation was studied by evaluating the effect of specific nonpeptide type 1 and type 2 AII receptor antagonists and the role of protein kinase-C (PKC). AII-induced DNA synthesis was significantly attenuated by two structurally dissimilar LO inhibitors, baicalein and phenidone. In addition, the LO product 12-hydroxyeicosatetraenoic acid (12-HETE) itself caused a significant increase in DNA synthesis, suggesting that the 12-LO pathway in part plays a role in AII-mediated mitogenesis. AII-induced proliferative responses were blocked by the type 1 AII receptor antagonist. Both AII- and 12-HETE-induced increases in DNA synthesis were markedly inhibited by two PKC blockers, staurosporine and sangivamycin. Further, both AII and 12-HETE could activate PKC by translocating it from the cytosol to the membrane fraction, as determined by Western immunoblotting. These results suggest that both 12-LO activation and protein kinase-C have an important role in AII-induced adrenal cell proliferation.


Constitutive expression of osteoclast-stimulating activity by normal clonal osteoblast-like cells: effects of parathyroid hormone and 1,25-dihydroxyvitamin D3.

Osteoblasts are known to produce osteoclast-stimulating activity (OSA). The aim of the current study was to relate the expression of OSA to the osteoblastic phenotype and examine its regulation by calciotropic hormones. The study was performed with the normal osteoblastic cell clone CRP 10/30 and the preosteoblastic clone CRP 4/7. OSA was determined with the well described isolated osteoclast pit assay, using sperm whale dentine as substrate. In contrast to previous studies, the assay was carried out at pH 7.36, rather than at pH 6.4 or 6.9. The results indicate that over 24 h, CRP 10/30 cells produce constitutively OSA, which compared to controls corresponds to an about 7-fold increase in resorption pits. There was considerably less activity expressed by either CRP 4/7 cells or fibroblasts. OSA proved to be heat labile, and its mol wt was estimated to be over 10 kilodaltons. While PTH-(1-34) did not influence the synthesis of OSA, the number of pits formed by osteoclasts incubated with medium conditioned by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-treated CRP 10/30 cells was increased 3-fold above baseline values. A similar increase was obtained with 1.25-(OH)2D3 added directly to CRP 10/30-conditioned medium. These results could not be duplicated with 1,25-(OH)2D3 added to either control medium or medium conditioned by CRP 4/7 cells or fibroblasts. The present study shows that normal clonal bone cells synthesize constitutively OSA, which is not regulated by PTH or 1,25-(OH)2D3. Furthermore, the results suggest that the synthesis of bone cell-derived OSA is limited to cells expressing the mature osteoblastic phenotype. Finally, CRP 10/30-conditioned medium appears to permit 1,25-(OH)2D3 to function on osteoclasts.


Biological mimicry of gonadotropin action by antiidiotypic antibodies to luteinizing hormone: characterization and biological properties.

High affinity polyclonal rabbit antibodies to ovine (o) pituitary LH [anti-oLH-immunoglobulin G (IgG) Ab1] were used to immunize young Southdown lambs. Their serum samples as well as those from controls receiving normal rabbit IgG were studied for the presence of anti-Ab1 antibodies. In RIAs using [125I]oLH and affinity-purified Ab1, sera from experimental sheep showed high activity, as expressed in oLH equivalents. These sera also showed ability to compete with [125I]oLH for binding to receptor on pig ovarian and testicular membranes. The antiidiotypic antibodies (Ab2) in experimental sheep sera were purified by successive affinity chromatography on immobilized rabbit normal IgG and immobilized oLH-IgG columns. Ab2-IgG eluted from the latter mimicked oLH in RIAs and RRAs. These purified Ab2 antibodies were also of a stimulatory type, because they elicited progesterone production in rat granulosa cells and collagenase-dispersed rat Leydig cells. This stimulatory action was counteracted by coincubation with anti-oLH-IgG, which would also terminate (oLH) hormone action in a similar manner. The Ab2 antibodies had no effect on oFSH RIA or on the binding of [125I]oFSH to pig ovarian receptors, indicating specificity with respect to LH antigenic structure and function. As can be expected from the choice of the immunogen (polyclonal anti-oLH-IgG), only a small percentage of the true Ab2 population could display biological mimicry of the original antigen (oLH). Their presence in circulation during 6-8 months had no effect on testicular size or body growth. The formation of Ab2 antibodies to rabbit anti-oLH-IgG was also demonstrated in male rats, but these were not purified. In this instance also there was no effect on testicular weight after 6 months of immunization. These results show the feasibility of producing antiidiotypic antibodies that stimulate gonadal function in a manner much like the pituitary gonadotropin (oLH).


Rat melanin-concentrating hormone messenger ribonucleic acid expression: marked changes during development and after stress and glucocorticoid stimuli.

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide first isolated from fish and rats. MCH may be involved in the control of the hypothalamic-pituitary-adrenocortical axis and, more generally, of specific goal-oriented behaviors and homeostatic functions in mammals. In this paper we examine 1) the cellular distribution of MCH gene transcripts in the rat central nervous system, 2) the changes in neuronal expression of MCH mRNA during rat development, and 3) the effects of stress and hormonal stimuli on rat MCH (rMCH) gene activity. Northern blot analysis and in situ hybridization histochemistry show that mature rMCH mRNA (1.0 kilobase) is very abundant in the zona incerta and the dorsolateral hypothalamus. While this is in agreement with previous peptide mapping by immunohistochemical techniques, a surprising new result is that a few clusters of rMCH mRNA-containing cells are found outside the hypothalamus, in the olfactory tubercle and the pontine tegmentum. Developmentally, rMCH mRNA is detected on embryonic day 18; its level increases gradually during early postnatal life and rises abruptly at weaning to reach a constant value in adult rats. In addition, striking variations in rMCH mRNA length occur during postnatal development and are found to be variations in the polyadenylate tail. Interestingly, this structural modification appears to be independent of the increase in rMCH mRNA levels. The regulation of rMCH mRNA expression by glucocorticoids and chronic stress is examined by Northern blot analysis. Chronic intermittent footshock stress causes a 58% or 29% decrease in rMCH mRNA content in the whole hypothalamus after a 1- or 3-day regimen, respectively. In contrast, the rMCH mRNA level returns to normal after a 7-day regimen. Two weeks after adrenalectomy (ADX) the whole hypothalamus rMCH mRNA content decreases 2.5-fold, but rises close to the control value 3 weeks after ADX. Dexamethasone administration 2 weeks after ADX not only reverses the fall in rMCH mRNA, it even provokes a slight increase (123% of control). No change in rMCH mRNA length is observed after chronic stress or ADX and dexamethasone injection. These results provide evidence for a negative regulation of rMCH gene expression by stress and suggest a major role for glucocorticoids in a positive feedback control of rMCH gene activity.


Thyrotropin-releasing hormone and lysine-bradykinin stimulate arachidonate liberation from rat anterior pituitary cells through different mechanisms.

TRH and lysine-bradykinin (Lys-bradykinin) increase PRL release and arachidonate liberation from anterior pituitary cells. We investigated whether the arachidonate liberation stimulated by TRH and Lys-bradykinin originates in pituitary lactotropes and whether these events are accomplished through similar mechanisms. Lys-bradykinin and TRH rapidly (0.5 min) increased the intracellular [3H]arachidonate content of rat anterior pituitary cells. Lys-bradykinin also increased [3H]arachidonate liberation and PRL release from lactotrope-enriched pituitary cells, but not from a pituitary cell preparation with a diminished number of lactotropes. In contrast, TRH increased [3H]arachidonate liberation from both lactotrope-enriched and lactotrope-diminished preparations; this increased [3H]arachidonate liberation stimulated by TRH in the lactotrope-diminished cells may originate in the thyrotropes. The effects of TRH and Lys-bradykinin on [3H]arachidonate and [14C]stearate liberation in perfused pituitary cells also were determined. Both secretagogues increased arachidonate and stearate liberation in a biphasic manner, characterized by a transient spike, followed by a lower magnitude wave of fatty acid release. The spike phase produced by Lys-bradykinin was more pronounced than that produced by TRH. The calcium dependence of TRH- and Lys-bradykinin-stimulated arachidonate liberation also was investigated. Cobalt and the low calcium medium containing ionomycin were used to block the secretagogue-induced increase in intracellular calcium concentrations. These conditions blocked TRH-stimulated arachidonate liberation, but only marginally decreased Lys-bradykinin-stimulated arachidonate liberation, indicating that the two peptides act through different mechanisms. Therefore, TRH stimulation of arachidonate liberation is linked to an increase in intracellular calcium. In contrast, Lys-bradykinin increases arachidonate liberation through a calcium-independent intracellular mediator. This calcium-independent increase in arachidonate liberation may involve the bradykinin receptor being coupled directly to a phospholipase, a G-protein that provides a link between the bradykinin receptor and the phospholipases that liberate arachidonate, or bradykinin-induced activation of a protein kinase-C that activates the phospholipases and subsequently liberates arachidonate.


Cellular localization and characterization of Glut 3 glucose transporter isoform in human brain.

In the present study we examined the expression and localization of Glut 3 in human brain using peptide-specific antisera. Glut 3 was expressed at 2-3 times higher levels in cerebral cortex from adult (n = 6) compared to that from neonatal infants (n = 4; P less than 0.05). However, similar levels of immunoreactive Glut 3 were present in cerebellum from adults (n = 6) and newborns (n = 4). Cellular localization of Glut 3 in adult (n = 5) and neonatal (n = 5) infant brains was undertaken by immunohistochemical analysis. Glut 3 was visible in the adult neuropil of the cerebral cortex; in certain cellular processes within the deeper cortical layers; in intravascular white cells, including monocytes, lymphocytes and granulocytes; and in microvascular endothelial cells. Neither the premature nor the mature newborn cerebral cortex exhibited Glut 3 labeling in the neuropil or microvasculature. In the cerebellum, given the stratified nature of the cellular arrangement, Glut 3 was more clearly and definitively noted in the cellular processes at all stages of development. Double labeling studies using neuronal (neurofilament) and astrocytic (glial fibrillary acidic protein) markers indicated that Glut 3 was primarily expressed in neurons. We conclude that Glut 3 is localized in many cellular components, including white blood cells in human brain. The prominent localization of Glut 3 to mature neuronal processes suggests an essential role for this transporter in regulating fuel requirements for dendritic and axonal traffic, thereby mediating neurotransmission. Further study is required to address the possibility that another as yet undefined glucose transporter isoform is expressed in other cell-specific regions of the brain.


A new, nongenomic estrogen action: the rapid release of intracellular calcium.

We have investigated the effects of steroids on the intracellular calcium ion concentration [Ca2+]i in chicken granulosa cells obtained from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye fura-2. The resting [Ca2+]i in these cells was 100 +/- 5 nM. There was an immediate (i.e. less than 5 sec) 4- to 8-fold increase in [Ca2+]i in all of the 76 cells examined after the addition of 10(-7) M estradiol-17 bdta. Estradiol-17 beta was effective between 10(-10)-10(-6) M. Estradiol-17 alpha, estrone, and estriol (10(-8)-10(-6) M) were as effective as estradiol-17 beta, but the progestins, pregnenolone, and progesterone, and the androgens, testosterone, androstenedione, or 5 alpha-dihydrotestosterone were ineffective at concentrations up to 10(-5) M. The prompt estradiol-17 beta-induced [Ca2+]i spike was not affected by incubating the cells in Ca(2+)-free medium containing 2 mM EGTA or by pretreating them with the Ca2+ channel blockers lanthanum (1 mM), cobalt (5 mM), methoxyverapamil (D600; 50 microM), or nifedipine (20 microM). The estrogen-triggered [Ca2+]i surge was also not affected by pretreating the cells with the conventional estrogen receptor antagonist tamoxifen (10(-5) M), or the RNA and protein synthesis inhibitors actinomycin D (1 microgram/ml) and cycloheximide (1 microgram/ml), but was abolished by pretreating the cells with inhibitors of inositol phospholipid hydrolysis, neomycin (1.5 mM) and U-73,122 (2.5 microM). The closely related, but inactive, compound U-73,343 (1 microM) did not affect the estrogen-triggered [Ca2+]i surge. Estradiol-17 beta (10(-7) M), but not progesterone (10(-5) M), also triggered a large [Ca2+]i surge in pig granulosa cells, which, like the [Ca2+]i surge in chicken granulosa cells, was almost immediate, transient, and unaffected by incubation in Ca(2+)-free medium or pretreatment with methoxyverapamil (D600; 50 microM), lanthanum (1 mM), or tamoxifen (10(-5)M). However, granulosa cells from immature rats primed with diethylstilbestrol or PMSG did not respond to estradiol-17 beta, even at concentrations as high as 10(-5) M, although they promptly generated a [Ca2+]i transient upon exposure to LHRH (10(-5) M). These results suggest that estrogens almost instantaneously trigger the release of Ca2+ from intracellular stores which may be mediated through phosphoinositide breakdown. The striking rapidity of this estrogen-induced internal Ca2+ mobilization is consistent with the activation of a cell surface receptor which is different from the conventional slowly acting, gene-stimulating nuclear estrogen receptor.


Identification of nuclear triiodothyronine receptors in the thymic epithelium.

Thymic epithelial cell physiology is known to be under neuroendocrine control. In particular, thyroid hormones modulate thymic hormone secretion by thymic epithelial cells in vivo and in vitro, thus suggesting the existence of specific receptors for those hormones in this component of the thymic microenvironment. Yet, thyroid hormone-binding sites have previously been detected only in crude thymus fractions and lymphocytes. We, thus, decided to search for T3 receptors in the thymic epithelium, by using an antinuclear T3 receptor monoclonal antibody. In situ immunohistochemical analysis of thymic frozen sections showed nuclear labeling of both lymphoid and nonlymphoid cells in the cortex and medulla. Moreover, in vitro studies using thymic epithelial cell lines and the so-called thymic nurse cells revealed a positive reaction in the chromatin, with nucleoli remaining negative. Immunoblot data clearly showed a single protein band of 57K reactive with the antinuclear T3 receptor antibody in murine thymus extracts as well as in the thymic epithelial cell lines. Lastly, in vitro treatment of these cells with T3 resulted in a transient, yet profound, down-modulation of the receptor. In conclusion, our findings provide molecular evidence that the action of thyroid hormones on thymic epithelium occurs via the typical 57K nuclear T3 receptors.


Effect of placement of dexamethasone adjacent to the ovine fetal paraventricular nucleus on adrenocortical steroid hydroxylase messenger ribonucleic acid.

The preterm rise in the concentration of ovine fetal plasma cortisol that initiates the events of parturition in sheep commences at approximately 125 days of gestational age (dGA; term = approximately 147 dGA). Concurrent with the rise in fetal plasma cortisol, adrenocortical steroid hydroxylase enzyme activity increases. The purpose of this study was 1) to quantitate changes in levels of mRNA for the steroid hydroxylases, 17-hydroxylase cytochrome P450 (P450(17)alpha), side-chain cleavage cytochrome P450 (P450scc), 11 beta-hydroxylase cytochrome P450 (P450(11)beta), and C21-hydroxylase cytochrome P450 (P450C21); and 2) examine the role of the fetal paraventricular nucleus (PVN) in the onset of adrenocortical steroid hydroxylase mRNA expression. Unperturbed fetuses were collected by cesarian section under halothane anesthesia at 105 (n = 4), 120 (n = 4), 126-128 (n = 4), and 136 dGA (n = 3); neonatal animals were collected within 2 h after birth. To examine the role of the fetal PVN in regulation of adrenocortical steroid hydroxylase mRNA expression, ovine fetuses were stereotaxically implanted bilaterally 2 mm lateral to the fetal PVN at 105-107 dGA with either cholesterol (n = 4) or dexamethasone (DEX; n = 3). Implanted fetuses were collected by cesarian section under halothane anesthesia at 126-128 dGA. mRNA for both cytochrome P450(17)alpha and P450scc declined 3-fold from 105 to 120 dGA (P less than or equal to 0.05), and then increased by 126-128 dGA compared to that on 120 dGA (P less than or equal to 0.05) and continued to increase through term. Cytochrome P450C21 increased at 126-128 dGA compared to that on 105 and 120 dGA (P less than or equal to 0.05) and remained elevated through term. Three distinct transcripts [approximately 6.2, 4.2, and 2.5 kilobases (kb)] were observed for cytochrome P450(11)beta; the 4.2-kb transcript was predominant. While total message for P450(11)beta declined over increasing gestational age, no differences were noted for the 4.2-kb transcript until after birth, when levels significantly declined (P less than or equal to 0.025). Placement of DEX adjacent to the fetal PVN prevented reemergence of expression of mRNA for P450(17)alpha and P450scc at 126-128 dGA, but had no effect on mRNA for P450C21 or P45011 beta. We conclude that mRNA for P450(17)alpha and P450scc undergoes a decline in expression concurrent with the previously described period of adrenal hyporesponsiveness from 105-126 dGA, followed by an increase in mRNA that accompanies the preterm rise in fetal plasma cortisol.(ABSTRACT TRUNCATED AT 400 WORDS)


Hypercortisolemia inhibits yohimbine-induced release of norepinephrine in the posterolateral hypothalamus of conscious rats.

Chronic hypercortisolemia attenuates yohimbine (YOH)-induced increments in plasma levels of the sympathetic neurotransmitter norepinephrine (NE). The present study used in vivo microdialysis to study the effects of hypercortisolemia on YOH-induced release of NE in the brain. Cortisol (25 mg/kg.day) or saline was infused sc into rats for 7 days via an osmotic minipump. Microdialysate and plasma concentrations of NE and its metabolites dihydroxyphenylglycol and methoxyhydroxyphenylglycol were measured before and after YOH (1 mg/kg, iv) administration in conscious animals, with microdialysate and plasma collections beginning 20-24 h after probe implantation. Chronic cortisol treatment resulted in attenuated NE, dihydroxyphenylglycol, and methoxyhydroxyphenylglycol responses in both microdialysate and plasma. The results indicate that YOH increases central neural as well as peripheral release, reuptake, turnover, and metabolism of NE and that hypercortisolemia suppresses these responses.


Elevated levels of endogenous pregnenolone fatty acid esters in follicular fluid high density lipoproteins support progesterone synthesis in porcine granulosa cells.

Fatty acid esters of pregnenolone (PREG-FA) are present at high levels within ovarian follicular fluid (FF), although no physiological function has yet been attributed to these pregnenolone metabolites. We demonstrate in the following study that PREG-FA are predominantly localized in association with high density lipoproteins (HDL) within FF at levels that surpass those found in plasma HDL by approximately 200-fold. Extensive treatment with dextran-coated charcoal revealed that although nonconjugated steroids could be removed very efficiently from whole fluid and HDL, little effect could be demonstrated on the PREG-FA levels present within the fluid or its HDL component. This observed resistance to charcoal stripping may reflect a steroid-protein interaction that is altogether different from the non-conjugated steroid-protein association. Both plasma and FF-derived HDL were equally effective in supporting PROG biosynthesis in porcine granulosa cell cultures under normal culture conditions, but when PROG synthesis was dramatically blocked by inhibiting the P450 side-chain cleavage enzyme complex with the P450 enzyme inhibitor ketoconazole, only HDL of FF origin was capable of restoring PROG synthesis. Similar cultures that received HDL of plasma origin, containing similar levels of total cholesterol but far less PREG-FA, showed no significant difference in PROG accumulation over control cultures under these conditions. Several reports have described the presence of an entity within FF that was shown to resist charcoal stripping and enhanced PROG synthesis in granulosa cell cultures. Our data suggest that this entity within FF may indeed be the high levels of endogenous PREG-FA. We, therefore, propose a role for PREG-FA as a potential substrate in the biosynthesis of PROG in porcine granulosa cells.


Dynamic regulation of follicle-stimulating hormone-beta messenger ribonucleic acid levels by activin and gonadotropin-releasing hormone in perifused rat pituitary cells.

Maintenance of FSH biosynthesis requires ongoing exposure to pulsatile GnRH. Recent data demonstrate that activin also stimulates FSH biosynthesis. We used a perifused pituitary system to examine regulation of FSH beta mRNA levels by pulsatile GnRH and activin. Hourly pulses of 10 nM GnRH increased FSH beta mRNA levels by 3-fold. In the same experiment, continuous infusion of 50 ng/ml activin elicited a 50-fold increase in FSH beta mRNA. This magnitude of response to activin in perifusion was unexpected, as only a 2.7-fold increase in FSH beta mRNA was observed when activin was administered to pituitary cells that were cultured in dishes. Since perifusion columns, unlike culture dishes, are exposed to a continuous supply of fresh medium, we examined the possibility that endogenous factors produced by pituitary cells cultured in dishes were stimulating the cells in a paracrine fashion, thereby precluding the full response to exogenously added activin. The kinetics of FSH beta mRNA expression were examined immediately after pituitary dispersion and at different times after culturing the cells in plates. FSH beta mRNA levels fell rapidly after dispersion to 8% of initial levels and remained low over 8 h. Thereafter, FSH beta mRNA levels increased slowly and exceeded initial levels by the second day of culture. In a parallel set of experiments, when medium conditioned by exposure to plated cells was applied to the perifusion system, FSH beta mRNA levels were selectively stimulated (6-fold). These data suggest the removal during dispersion and subsequent accumulation in culture of pituitary-derived factors that are important for the maintenance of FSH beta mRNA levels. We conclude that activin plays a greater role in the regulation of FSH beta mRNA levels than was suggested by previous experiments employing static culture systems in which autocrine or paracrine stimulation may have obscured the effects of exogenously added activin.


Sexual dimorphism in regulation of type II corticosteroid receptor immunoreactivity in the rat hippocampus.

To determine whether there are sex differences in the distribution of type II corticosteroid receptor-immunoreactive (type II-ir) cells in the rat hippocampus, we carried out a quantitative morphometric immunocytochemical study using a mouse monoclonal antibody, BUGR2. We report that in adrenally intact male and female rats, high densities of cells with nuclear type II-ir were observed in the pyramidal layer of field CA1 and the granular layer of the dentate gyrus. In intact males very few cells, presumably glia, in the stratum oriens showed type II-ir. In contrast, in females, interneurons with diffuse or cytoplasmic type II-ir were observed in the stratum oriens of CA1 and CA3. There were also sex differences in the regulation of type II-ir by corticosterone, the predominant glucocorticoid, and female sex steroids. In male rats the density of cells with nuclear type II-ir in all parts of Ammon's horn and the dentate gyrus was decreased significantly after adrenalectomy (adx). In contrast, in females such reductions were observed only in the pyramidal layer of CA1 and the granular layer of the dentate gyrus. In both sexes, cells with intense diffuse or mainly cytoplasmic type II-ir were observed in the pyramidal layer and stratum oriens after adx. The loss of nuclear type II-ir in the hippocampus of adx females was not affected significantly by ovariectomy. In adx males, nuclear Type II-ir was restored in CA1 and the dentate gyrus after treatment with corticosterone or progesterone. Cells in CA3 were, however, unresponsive to treatment with either hormone. In contrast, in adx females, treatment with either corticosterone or progesterone restored nuclear type II-ir to cells in all regions of the hippocampus. In both adx males and females, cytoplasmic type II-ir observed in some cells in the pyramidal layer and stratum oriens, was abolished completely by corticosterone, and partially by progesterone treatment. In both adx males and females, estradiol treatment did not affect significantly the pattern of type II-ir. Sex differences in the distribution of type II-ir interneurons in intact rats and the regulation of the intracellular location of type II-ir of adx rats by corticosterone and progesterone, may be important determinants of sex differences in the modulation of hippocampal function by glucocorticoids.


Increases in cellular sodium concentration by arginine vasopressin and endothelin in cultured rat glomerular mesangial cells.

The present study was undertaken to determine whether arginine vasopressin (AVP), angiotensin-II, and endothelin (ET) increase the cellular sodium concentration ([Na+]i) in cultured rat glomerular mesangial cells. [Na+]i was measured using the fluorescence indicator dye sodium-binding benzofuran isophthalate. These three vasoconstrictor hormones increased cellular free calcium ([Ca2+]i) and [Na+]i in a dose-dependent manner ([Na+]i: basal, 11.5; 10(-7) M AVP, 20.5; 10(-7) M angiotensin-II, 13.8; and 10(-7) M ET, 21.2 mM). The mobilization of [Ca2+]i was faster than that of [Na+]i. The AVP-induced increase in [Na+]i was completely blunted by the potent V1 antagonist d(CH2)5Tyr(Me)AVP. Vasoconstrictor hormones produced a biphasic cellular pH (pHi) change, characterized by a transient acidification, followed by a sustained alkalinization. The Ca(2+)-free condition markedly reduced AVP- and ET-induced increases in [Ca2+]i and [Na+]i and biphasic changes in pHi. In the Na(+)-free state, the hormonally mobilized [Ca2+]i was significantly enhanced. Basal [Na+]i decreased to below 3 mM, and there was little increase in [Na+]i after the addition of vasoconstrictor hormones, suggesting that the source of [Na+]i is extracellular space. Only early acidification was obtained in the absence of a sustained alkalinization. The [Na+]i mobilization was closely related to the biphasic change in pHi. These results indicate that AVP, ET, and angiotensin-II increase [Na+]i in glomerular mesangial cells, and that the early mobilization of [Na+]i depends on Na+/Ca2+ exchange, and the sustained phase depends on Na+/H+ exchange. The hormonally mobilized [Ca2+]i is essential for the activation of Na+/H+ exchange, and an increase in [Na+]i is suggested to play an important role in cellular alkalinization.


Calcium homeostasis in growth hormone (GH)-secreting adenoma cells: effect of GH-releasing factor.

Human GH-secreting tumors are heterogenous regarding their basal secretory activity and response to GH-releasing factor (GRF). We have investigated whether such different secretory properties could be accounted for by alterations of intracellular mechanisms occurring at the calcium level. Basal free intracellular calcium concentrations ([Ca2+]i) and Ca2+ responses to GRF were studied in single cells cultured from fragments of five GH-secreting pituitary adenomas. We used the microspectrofluorimetric method and indo-1 as the fluorescent probe. The cell populations cultured from the tumors of patients A and C showed increased hormone secretion in response to GRF in vitro, whereas cultures from patients B, D, and E were unresponsive to the peptide. Basal [Ca2+]i measured in the five cell populations ranged from 82 +/- 18 to 118 +/- 27 nM. A 10-sec application of 10 nM GRF induced an increase in [Ca2+]i in 60% and 54% of A and C cells, respectively. In the nonresponsive cell populations, the number of calcium responses to GRF was lower, 26% (B cells), 5% (D cells), and 10% (E cells). Two principal responses types were observed: 1) an initial increase in [Ca2+]i, followed by a sustained plateau phase lasting for more than 200 sec; and 2) a monophasic peak of increased [Ca2+]i lasting approximately 1 min before returning to baseline levels. GRF responses were totally suppressed in the absence of Ca2+ ions in the external medium. Sixteen to 30% of the cells cultured from four of the five tumors showed spontaneous fluctuations of [Ca2+]i. These spontaneous Ca2+ transients were suppressed in Ca(2+)-free medium. The number of cells exhibiting such Ca2+ transients decreased with time in culture. Basal hormone secretion was higher in cultures from patient D, in which no spontaneous Ca2+ transients were observed in any of the 72 studied cells, and in cultures from patients E, in which only 16% of cells were spontaneously active. We conclude that 1) in human responsive somatotrophs, the involvement of Ca2+ in GRF stimulus-secretion coupling mechanisms is apparently similar to that described in somatotrophs of other species; 2) the lack of a secretory response to GRF observed in some tumors may result from impairment of Ca2+ responsiveness in either cell recruitment or response amplitude and/or duration; and 3) spontaneous rhythmic Ca2+ activity is apparently dissociated from basal hormone secretion in some of these tumor cells.


Characterization of angiotensin-II receptor subtype on bovine thecal cells and its regulation by luteinizing hormone.

In this study the primary objectives were to localize angiotensin-II (AII) receptors on specific ovarian cells and determine whether these receptors are regulated by LH. AII receptor analysis, carried out using membrane fractions prepared from isolated thecal, granulosa, and luteal cells from bovine ovary, revealed that [125I]AII-binding sites were present only on thecal cells. The Kd and binding capacity were determined to be 0.29 +/- 0.08 nM and 66.9 +/- 8.1 fmol/mg membrane protein (mean +/- SEM from three experiments, each with duplicate determinations), respectively. None of the peptides unrelated to AII affected the binding of [125I]AII. Unlabeled AII, saralasin, and AIII were equipotent (IC50, approximately 5 nM for all three peptides) in competing with the radioligand. However, the binding affinity for AI was less by almost 2 log units. Using AII receptor subtype-specific nonpeptide antagonists, Losartan [a selective antagonist for the type 1 AII (AT1) receptor] and PD 123319 [a selective antagonist for the type 2 AII (AT2) receptor], AII receptors on thecal cells could be classified pharmacologically as AT2-type receptors. The IC50 determined from the competitive binding inhibition experiments for the various unlabeled competing substances were 5 nM, 20 nM, 200 nM, and 200 microM with respect to AII, p-amino-phenylalanine-AII, PD123319, and Losartan, respectively. Thecal cells cultured in a serum-free medium also expressed AII receptors, which could be up-regulated by LH or 8-bromo-cAMP in a dose-dependent manner. The number of AII receptors on thecal cells nearly doubled when the cells were cultured in the presence of 100 ng/ml LH, with little change in their Kd value. This increase in the number of AII receptors was inhibitable by a protein synthesis inhibitor, cycloheximide. In summary, we have demonstrated that in the bovine ovary, AII receptors belonging to AT2 subclass are predominantly expressed on thecal cells, and these receptors can be up-regulated by LH via a cAMP-dependent mechanism. Thus, the bovine thecal cells in primary culture can potentially become a useful in vitro system to study the mechanism of regulation of AII receptor induction as well as the so far unknown function of this class of receptor.


Increased plasma concentrations, hypothalamic content, and in vitro release of arginine vasopressin in inflammatory disease-prone, hypothalamic corticotropin-releasing hormone-deficient Lewis rats.

The susceptibility of Lewis (LEW/N) rats to severe inflammatory disease has been causally associated with subnormal responsiveness of their hypothalamic CRH-secreting neurons and, consequently, their hypothalamic-pituitary-adrenal axis to several stimulatory neurotransmitters and inflammatory cytokines. In the present study we investigated in this strain the secretory dynamics of another major activator of pituitary ACTH secretion, arginine vasopressin (AVP). To accomplish this, we evaluated the circadian plasma concentrations and circadian and glucocorticoid-induced changes in hypothalamic content and in vitro release of AVP in 8- to 10-week-old female LEW/N rats and compared these measurements to those obtained in parallel from age- and sex-matched histocompatible, inflammatory disease-resistant Fischer (F344/N) rats. Plasma concentrations and hypothalamic content and in vitro release of AVP were significantly elevated in LEW/N compared to F344/N rats in both the morning and evening. These indices of higher AVP secretion in LEW/N than in F344/N rats were also present after chronic dexamethasone treatment. These findings suggest increased AVP production and release in LEW/N rats, perhaps representing an adaptive compensation for insufficient CRH and glucocorticoid secretion. The high levels of circulating AVP might contribute to the excessive inflammatory responses of these animals.


Lactoferrin expression in the mouse reproductive tract during the natural estrous cycle: correlation with circulating estradiol and progesterone.

Lactoferrin (LTF), an iron-binding glycoprotein present in most exocrine secretions and in the secondary granules of polymorphonuclear leucocytes (PMN), is regulated by estrogen in the mouse reproductive tract. We investigated the expression of LTF mRNA and protein during the natural estrous cycle to increase our understanding of how this uterine secretory protein is regulated under physiological conditions. There was a positive correlation between LTF mRNA expression in the genital tract and serum estradiol (E2) concentrations. When E2 peaked in proestrus, LTF mRNA and protein were expressed in the uterus; however, during metestrus, when both E2 and progesterone levels were high, LTF mRNA was expressed, while LTF protein was decreasing. LTF protein expression may be hindered by progesterone or some other local factor in the endometrial epithelium after ovulation. Immunohistochemistry demonstrated two distinct staining patterns for LTF in the vaginal and endometrial epithelium. In one staining pattern, the colorimetric reaction was noted over the cytoplasm, and in the other, the nuclear region stained more intensely. This suggests the possibility that in addition to its known role as a secretory protein, LTF may be transported to the nucleus, serving an autocrine role. Our results also indicated that LTF protein is a useful marker for tracking PMN. Nonproliferating epithelial cells in the vagina and endometrium may synthesize chemotactic and/or adhesion molecules for PMN.


Sex difference in the effect of mating on c-fos expression in luteinizing hormone-releasing hormone neurons of the ferret forebrain.

The pulsatile secretion of LH was previously found to rise in female ferrets after receipt of an intromission, whereas in males that achieved an intromission, both LH and testosterone secretion were either reduced or unchanged. We sought to determine whether this sexually dimorphic pattern of LH secretion reflects a sex difference in the effect of mating on the activity of forebrain neurons that secrete LHRH. Immunocytochemical methods were used to localize the nuclear protein product of the immediate early gene, c-fos, as an index of increased neuronal activity after mating. Nuclear FOS immunoreactivity (FOS-IR) was monitored in LHRH-IR neurons as well as other non-LHRH forebrain neurons. In confirmation of previous reports, LHRH-IR perikarya in ferrets of both sexes were located medially along the base of the brain at rostral, medial, and caudal levels of the preoptic-hypothalamic continuum. In each of these regions a significantly higher percentage of LHRH-IR neurons was colabeled with nuclear FOS-IR in mated than in unpaired females. By contrast, an equivalent low percentage of LHRH-IR neurons was colabeled with FOS-IR in mated and unpaired male ferrets. Significantly more FOS-IR neurons (not colabeled with LHRH) were detected in the bed nucleus of the stria terminalis, the medial preoptic area, the dorsal-medial hypothalamus, and the medial amygdala (MA) of mated vs. unpaired females. By contrast, mating significantly augmented FOS-IR only in the MA of male ferrets. The results suggest that the sexually dimorphic pattern of LH secretion that occurs in ferrets after mating reflects a selective activation of LHRH neurons in the female forebrain. This sex-specific increase in the responsiveness of LHRH neurons to mating may depend on input from a limbic circuit which includes the medial amygdala, bed nucleus of the stria terminalis, and medial preoptic area.


Effect of human recombinant mullerian inhibiting substance on isolated epithelial and mesenchymal cells during mullerian duct regression in the rat.

The effect of human recombinant Mullerian Inhibiting Substance (MIS) on the regression of the Mullerian duct (MD) of female rat fetuses was examined in vitro to determine whether MIS acts on MD epithelium and/or mesenchyme at the critical periods of sexual differentiation. Urogenital ridges (URs) of female rat fetuses at 14.5- to 18.5-days of gestation (plug day = 0) were cultured for 3 days with or without recombinant human MIS in CMRL 1066 medium with 10% female fetal calf serum. In URs from 14.5- and 15.5-day-old fetuses, the cranial portion of the MD regressed almost completely during the 3-day culture period in the presence of MIS, whereas the caudal half to third of the MD remained intact but tapered to a fine point cranially. MDs survived in URs from 16.5-day-old fetuses cultured in the presence of MIS except that the cranial portion of the MDs was deformed. MIS did not elicit regression of MDs in URs obtained from 17.5- and 18.5-day-old fetuses, but instead caused the MD epithelium to form bulges projecting into the mesenchyme. MD epithelium at 15.5-days of gestation was separated from the surrounding UR mesenchyme, and both components (MD epithelium and mesenchyme) were cultured separately for 3 days in the presence or absence of MIS. Both epithelial and mesenchymal cells survived in the presence or absence of MIS. MD epithelium formed typical epithelial colonies, whereas UR mesenchyme spread as fibroblastic cells. Analysis of labeling index after incorporation of [3H] thymidine demonstrated that MD epithelial DNA synthesis was not influenced by MIS. In contrast, mesenchymal labeling index was reduced significantly by MIS. This effect of MIS on UR mesenchyme in conjunction with earlier histological observations of mesenchymal condensation during MD regression and an absence of direct effects of MIS on the epithelium suggests that MIS elicits its effect on the MD epithelium via the surrounding mesenchyme.


Multiple forms of affinity-labeled estrogen receptors in rat distinct pituitary cells.

The presence of multiple monomeric forms has been described for estrogen receptor (ER) in different target tissues. Using [3H]tamoxifen aziridine ([3H]TA) to covalently label ER and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to analyze labeled products, ER forms were investigated in pituitary cytosol and purified nuclei from male rats. ER forms were also compared in cellular extracts from gonadotrope-enriched populations (GP), prepared using the fast method of centrifugal elutriation, and from lactotrope-somatotrope fractions (LSP), obtained by sequential use of both elutriation and Percoll gradient sedimentation. A major labeled protein of 60,000-65,000 mol wt (M(r)) and a minor species of 50,000-55,000 M(r) were found in the pituitary cytosol and nuclear extracts covalently labeled with [3H]TA. The same results were obtained after ER covalent labeling from cellular extracts or intact dispersed cells. In gonadotrope-enriched cell population (greater than or equal to 50% LH-immunoreactive cells), the 65,000 M(r) species is the single unique ER form; in the LSP (80% PRL- and GH-immunoreactive cells), the major TA-labeled species is the 50,000 M(r) form, while the 65,000 M(r) ER is hardly detectable. Thus, the prevalence of 65,000 M(r) protein in the initial cell population can be explained by the higher number of binding sites per gonadotrope than per lactotrope cell. In conclusion, ER heterogeneity is demonstrated in pituitary cell populations. The source of this heterogeneity could be due to 1) different ER mRNAs according to cell type, or 2) a specific posttranslational processing, such as proteolytic activity within lactotrope cells.