Differential ontogenetic patterns of in vitro desensitization to GHRH in fetal and neonatal anterior pituitary.

The aim of this study was to assess the ontogenetic changes in vitro in both the responsiveness of anterior pituitary tissue to growth hormone-releasing hormone (GHRH) and the critical role of GHRH in the long-term regulation of pulsatile GH secretion during perinatal porcine life. A superfusion system was used to apply three consecutive 10-min pulses of GHRH (the first of 1 nM and the other two of 10 nM) for 3 consecutive days in pituitary glands isolated from fetal (95- and 110-day) and neonatal (12-day) male pigs. In fetuses, total GHRH-induced GH release decreased progressively over the 3 days. However, in neonates, GH did not decrease until day 3, but remained higher than in fetuses. When each GH pulse was assessed individually, fetuses showed a similar pattern. GH secretion induced by the first GHRH pulse on days 1 and 2 was lower than that induced by the second and third pulses. By day 3, GH release lowered dramatically after all pulses. In contrast, in neonates no differences were observed among the GH levels induced by the three GHRH pulses at any day, although day 3 showed lower GH rates. In conclusion, during perinatal development, a desensitizing effect to long-term repetitive GHRH pulses was observed in both fetuses and neonates, but this effect was delayed in neonates. Thus, the capacity of somatotrope cells to maintain GH response to GHRH seems to be developmentally regulated during perinatal stages. Furthermore, the frequency of GHRH pulses, rather than the concentrations, might be a key factor to elicit desensitization.

Pneumocystis jiroveci genotypes in the Spanish population.

This study describes the genotype distribution of Pneumocystis jiroveci in 79 respiratory samples obtained from 15 patients with acquired immunodeficiency syndrome (AIDS) with P. jiroveci pneumonia and 64 human immunodeficiency virus-negative subjects with different chronic pulmonary diseases. The genotyping was based in analysis of 2 independent genetic loci: the mitochondrial large subunit ribosomal RNA (mt LSU rRNA) fragment (assessed by direct sequencing) and the gene for dihydropteroate synthase (DHPS; assessed by restriction fragment-length polymorphism). The mt LSU rRNA analysis revealed the presence of 3 different polymorphisms for both populations. The major genotype, 85C/248C, was found to be significantly higher in patients with AIDS and P. jiroveci pneumonia than in patients with pulmonary disease. The rate of genotypes 85A/248C and 85T/248C was similar in both groups. The analysis of DHPS genotypes assesses the prevalence of its 4 possible genotypes, with 35.5% of genotypes related to sulfa resistance. The data suggest a common source of infection between both groups.

Comparison of amplicor, in-house polymerase chain reaction, and conventional culture for the diagnosis of tuberculosis in children.

A total of 251 clinical specimens (235 gastric aspirates and 16 bronchoalveolar lavages) from 88 children were prospectively tested in a blinded manner for the presence of Mycobacterium tuberculosis complex, by use of the Amplicor M. tuberculosis test and by means of in-house polymerase chain reaction (PCR). The results were compared with those obtained by conventional culture and by direct microscopy. All of the children underwent extended follow-up to verify or exclude the clinical diagnosis of tuberculosis. The results of the different tests, when compared to the final clinical diagnosis, were a sensitivity of 60% and a specificity of 96.8% for in-house PCR, 44% and 93.7% respectively for the Amplicor test, 44% and 100% for mycobacterial culture and 12% and 100% for microscopy. Amplicor tests presented false-positive findings in children without tuberculous infection. We conclude that both in-house PCR and the Amplicor test are rapid methods that can be helpful for difficult or urgent diagnosis of tuberculosis in children. However, efforts should be aimed toward improvement of the sensitivity and specificity of an easy-to-use PCR kit.

Ototoxicity caused by aminoglycosides is ameliorated by melatonin without interfering with the antibiotic capacity of the drugs.

The production of free radicals seems to be involved in the mechanisms of ototoxicity. Aminoglycosides produce ototoxicity, which can be determined through distortion product otoacoustic emissions (OAEs) that measure the activity of the outer hair cells of the organ of Corti. An ototoxic chart was obtained in rats using gentamicin or tobramycin. Together with this treatment, the animals ingested melatonin in the drinking water, or melatonin was injected subcutaneously or intramuscularly. The distortion product OAEs were determined over a prolonged period of time for each of the groups. The effect of melatonin on the antibiotic capacity of the aminoglycosides used was also studied. Antibiograms inoculated with Escherichia coli or Pseudomonas aeruginosa and treated with gentamicin or tobramycin in the presence or absence of melatonin at quantities from pharmacological to physiological doses were performed. The ototoxicity produced by gentamicin and tobramycin was maximal from days 3 to 5 post-treatment, returning to normal values in 2 wk. When melatonin was present, the recovery was at day 5 post-treatment, independently of the means of administration of the pineal product. The antibiograms showed that melatonin had no effect on the antibiotic capacity. It is concluded that the ototoxicity caused by gentamicin and tobramycin is ameliorated by melatonin and that the pineal hormone does not interfere with the antibiotic capacity of these antibiotics.

Diagnosis of tuberculosis in children using a polymerase chain reaction.

We investigated the value of the polymerase chain reaction (PCR) in the diagnosis of active tuberculosis in children and evaluated the relationship between PCR results in children with tuberculous infections and mediastinal adenopathies detected by computerized tomography (CT-Scan). This was a controlled, blinded, prospective study comparing nested PCR, mycobacterial cultures and the clinical diagnosis based on 350 clinical specimens from 117 children referred for evaluation of suspected pulmonary tuberculosis. All children with tuberculous infection but without active disease underwent a chest CT-scan to detect the presence of mediastinal adenopathies not evident on chest x-ray. The sensitivity of PCR was 56.8% in children with clinically active disease (culture: 37.8%; smears: 13.5%). A major advantage of PCR over cultures was noted when there was no parenchymal involvement on chest radiograph and when the patient was undergoing anti-tuberculous treatment. There were nine specimens with false-negative PCR results due to the presence of amplification reaction inhibitors. PCR was positive in five children with tuberculous infection without active disease and these children presented mediastinal adenopathies on the CT-scan that were not evident on chest radiography. There were no false-positive PCR results in the control groups of children. We conclude that nested PCR is a rapid and sensitive method for the early diagnosis of tuberculosis in children. It is especially useful when the diagnosis of active tuberculosis is difficult. In our study children with tuberculous infection without apparent disease who have positive PCR results have mediastinal adenopathies on CT-scan.

Murine typhus as a common cause of fever of intermediate duration: a 17-year study in the south of Spain.

BACKGROUND: Fever of intermediate duration (FID), characterized by a febrile syndrome lasting from 7 to 28 days, is a frequent condition in clinical practice, but its epidemiological and etiologic features are not well described. Murine typhus (MT) is a worldwide illness; nevertheless, to our knowledge, no studies describing its epidemiological and clinical characteristics have been performed in the south of Spain. Also, its significance as a cause of FID is unknown. OBJECTIVE: To determine the epidemiological features, clinical characteristics, and prognosis of MT and, prospectively, its incidence as a cause of FID. DESIGN: Prospective study of cases of MT over 17 years (1979-1995) and of all cases of FID treated in a tertiary teaching hospital in Seville, Spain. RESULTS: One hundred and four cases of MT were included, and MT was the cause in 6.7% of 926 cases of FID. Insect bites were reported in only 3.8% of the cases of MT previous to the onset of illness. Most cases (62.5%) occurred in the summer and fall. A high frequency of rash (62.5%) was noted. Arthromyalgia (77%), headache (71%), and respiratory (25%) and gastrointestinal (23%) symptoms were also frequent. Laboratory findings were unspecific. Organ complications were uncommon (8.6%), but they were severe in 4 cases. The mean duration of fever was 12.5 days. Cure was achieved in all cases, although only 44 patients received specific treatment. CONCLUSIONS: Murine typhus is prevalent in the south of Spain and is a significant cause of FID. Clinical signs are benign, but some patients may develop severe complications. A high degree of clinical suspicion is required for diagnosis.

Growth hormone (GH)-releasing factor differentially activates cyclic adenosine 3',5'-monophosphate- and inositol phosphate-dependent pathways to stimulate GH release in two porcine somatotrope subpopulations.

Somatotropes comprise two morphologically and functionally distinct subpopulations of low (LD) and high (HD) density cells. We recently reported that GRF induces different patterns of increase in the cytosolic free Ca2+ concentration in single porcine LD and HD somatotropes, which for LD cells required not only Ca2+ influx but also intracellular Ca2+ mobilization. This suggested that GRF may activate multiple signaling pathways in pig LD and HD somatotropes to stimulate GH secretion. To address this question, we first assessed the direct GRF effect on second messenger activation in cultures of LD and HD cells by measuring cAMP levels and [3H]myo-inositol incorporation. Secondly, to determine the relative importance of cAMP- and inositol phosphate (IP)-dependent pathways, and of intra- and extracellular Ca2+, GRF-induced GH release from cultured LD and HD somatotropes was measured in the presence of specific blockers. GRF increased cAMP levels in both subpopulations, whereas it only augmented IP turnover in LD cells. Accordingly, adenylate cyclase inhibition by MDL-12,330A abolished GRF-stimulated GH release in both subpopulations, whereas phospholipase C inhibition by U-73122 only reduced this effect partially in LD cells. Likewise, blockade of Ca2+ influx with Cl2Co reduced GRF-stimulated GH secretion in both LD and HD somatotropes, whereas depletion of thapsigargin-sensitive intracellular Ca2+ stores only decreased the secretory response to GRF in LD cells. These results demonstrate that GRF specifically and differentially activates multiple signaling pathways in two somatotrope subpopulations to stimulate GH release. Thus, although the prevailing signaling cascade employed by GRF in both subpopulations is adenylate cyclase/cAMP/extracellular Ca2+, the peptide also requires activation of the phospholipase C/IP/intracellular Ca2+ pathway to exert its full effect in porcine LD somatotropes.

Direct effects of growth hormone (GH)-releasing hexapeptide (GHRP-6) and GH-releasing factor (GRF) on GH secretion from cultured porcine somatotropes.

Growth hormone (GH)-releasing hexapeptide (GHRP-6) belongs to the expanding family of synthetic GH secretagogues (GHSs). Previous studies have shown that non-peptidyl GHRP-6 analogues stimulate GH release in vivo in pigs, and interact synergistically with GH-releasing factor (GRF), but its direct effects on porcine somatotropes have not been addressed hitherto. In the present study, we have evaluated the response of cultured porcine pituitary cells to GHRP-6, and its interaction with GRF and somatostatin (SRIF). Secretory response of somatotropes was assessed by using two distinct techniques. GH released by monolayer cell cultures was evaluated by enzyme immunoassay, whereas that secreted by individual somatotropes was measured by immunodensitometry using a cell blotting assay. Our results demonstrate that both GHRP-6 and GRF stimulated GH release from monolayer cultures at doses equal to or above 10(-9) M. Use of cell immunoblot assay demonstrated that, like GRF, the hexapeptide acts directly upon porcine somatotropes to exert its action. Moreover, regardless of the technique applied, combined administration of GHRP-6 (10(-6) or 10(-9) M) and GRF (10(-8) M) resulted in an additive, but not synergistic, stimulatory GH response. Finally, SRIF (10(-7) M) inhibited the stimulatory effect of GHRP-6 alone or in combination with GRF. These results indicate that GHRP-6 directly and effectively stimulates GH secretion from porcine somatotropes in vitro, and acts additively when coadministered with GRF. Therefore, the synergistic stimulatory effect of GHSs and GRF reported in vivo in this species might require additional factors that are lacking in the in vitro situation.

Growth hormone-releasing factor mobilizes cytosolic free calcium through different mechanisms in two somatotrope subpopulations from porcine pituitary.

Porcine somatotropes can be separated by Percoll density gradient centrifugation into low (LD) and high density (HD) subpopulations that differ ultrastructurally and functionally. Here, we report the effects of growth hormone-releasing factor (GRF) on the cytosolic free calcium concentration ([Ca2+]i) of single LD and HD somatotropes. Resting [Ca2+]i in LD somatotropes was 2-fold higher than in HD cells. GRF induced [Ca2+]i increases in a similar percentage of somatotropes from both subsets. However, amplitude and kinetics of the responses were markedly different. In all responsive LD somatotropes, GRF evoked a rapid initial peak followed by a sustained plateau (plateau-type response). Blockade of extracellular Ca2+ entry by 3 mM EDTA, 2 mM CoCl2, or 100 microM verapamil completely abolished the plateau phase without affecting the initial Ca2+ spike. Conversely, only the plateau phase was preserved in thapsigargin (TG)-treated LD cells. The vast majority of GRF-responsive HD somatotropes exhibited a transient [Ca2+]i peak that returned gradually to baseline (transient-type response). This response was completely blocked by removal of extracellular Ca2+, whereas TG treatment had no effect. Taken together, our results indicate that the response of LD somatotropes to GRF depends on mobilization of Ca2+ of both extra- and intracellular origin, whereas that of HD somatotropes seems to be exclusively dependent on extracellular Ca2+ entry through L-type voltage sensitive Ca2+ channels (VSCC). These findings are the first to demonstrate a differential effect of GRF on Ca2+ mobilization in two somatotrope subpopulations, and suggest the existence of differences in the GRF receptor(s) expressed in each subpopulation and/or in the intracellular signalling pathways activated upon GRF binding.

Unexpected high prevalence of hepatitis C virus genotype 4 in Southern Spain.

BACKGROUND/AIMS: A unusually high rate of HCV-infected individuals in whom the HCV genotype cannot be ascertained by means of single PCR and LIPA procedures has recently been reported in our area. The aim of the present study was to investigate the epidemiological, clinical and molecular characteristics of these patients. METHODS: Cross-sectional study. Eighty anti-HCV-positive patients with chronic liver disease, 45 (56.25%) of them intravenous drug users, were included. HCV genotyping was carried out in all patients using commercial single PCR and LIPA procedures. Samples where no HCV RNA amplification and/or indeterminate HCV genotype were found were also tested by means of a nested PCR. HCV viral load was measured in all patients. RESULTS: HCV genotyping was not achieved in 23 (28.75%) individuals. No amplification of HCV RNA was found in 19 of them, and in four other cases the LIPA procedure did not allow identification of a distinct HCV genotype. After the use of nested PCR+LIPA, it was found that the HCV genotype 4 was found in 11 of those 23 individuals (47.82%). Ten of these 11 HCV genotype 4-harboring individuals were intravenous drug users. The HCV viral load was lower in HCV genotype 4-harboring individuals than in those whom the genotypes 1, 2 or 3 were found (p<0.001). CONCLUSIONS: A high rate of HCV genotype 4-harboring cases has been found among HCV-infected individuals in Southern Spain. Had only single PCR been used, these individuals could have been wrongly regarded as non-viremic.

Control of growth hormone secretion from porcine fetal and neonatal pituitary tissue in vitro by growth hormone-releasing hormone, somatostatin, and insulin-like growth factor.

The physiological mechanism underlying the enormously elevated fetal plasma GH concentrations in mammalian species is not well understood. We postulated that a decreased sensitivity to the inhibitory effects of somatostatin (SRIH) and insulin-like growth factor-1 (IGF-1) at the pituitary level during porcine fetal life might be one factor in the high plasma GH levels. Therefore, the acute effects of GH-releasing hormone (GHRH), SRIH, and IGF-1 on GH release from the porcine anterior pituitary (AP) were studied using a perifusion system. AP tissue pieces from male and female fetuses (95 days postcoitum) and piglets (10-12 days postpartum) were perifused (at least 6 replicates per treatment and sex) at a rate of 0.1 ml/min and fractions were collected at 10-min intervals. Tissue was subjected to various treatments in random order: a 10-min GHRH pulse (1 nM) with or without concomitant 40-min exposure to SRIH (1, 10 or 100 nM) or IGF-1 (1 or 10 nM) or exposure to SRIH or IGF-1 alone (10 nM each). None of the treatments revealed sex differences, therefore data from males and females were pooled. Exposure to GHRH resulted in a rapid stimulatory GH response both in fetuses (from 61.1 +/- 4.1 to 125.6 +/- 4.7 ng GH.80 min-1.mg-1 AP, p < 0.05) and neonates (from 58.1 +/- 5.3 to 104.6 +/- 4.5 ng GH.80 min-1.mg-1 AP, p < 0.05). Exposure to the lowest dose of SRIH or IGF-1 (1 nM) during GHRH pulse inhibited (p < 0.05) the GH response to GHRH in neonates (74.3 +/- 3.9 and 72.5 +/- 6.3 ng GH.80 min-1.mg-1 AP, respectively), but not in fetuses (115.9 +/- 5.8 and 118.7 +/- 5.4 ng GH.80 min-1.mg-1 AP, respectively). When high doses of SRIH (10 or 100 nM) or IGF-1 (10 nM) were used, the GHRH-induced GH release was totally blocked in neonates (p < 0.05). However, in fetuses the GH response was inhibited only during the first 40 min after GHRH, and a rebound or delay effect occurred during the next 40 min. Irrespective of these general findings, some individual profiles from fetuses (4 profiles) showed that 1 nM SRIH or IGF-1 could inhibit GHRH-induced GH secretion. In contrast, individual profiles from neonates indicated that high doses of SRIH (7 profiles) or IGF-1 (5 profiles) were unable to block GH release stimulated by GHRH. On the other hand, a paradoxical stimulation of GH was observed during treatment with SRIH alone in three individual profiles from fetuses. These data confirm our hypothesis that the highly elevated plasma GH levels in the porcine fetus in vivo (compared to neonates) may be explained, at least in part, by a decreased sensitivity to the inhibitory effects of SRIH and IGF-1 at the pituitary level during fetal life. Results from some profiles indicated individual variations in the maturation of the GH control system, and moreover, even a 'paradoxical' enhancement of GH release in some fetal GH profiles.

Human immunodeficiency virus infection modifies the natural history of chronic parenterally-acquired hepatitis C with an unusually rapid progression to cirrhosis.

BACKGROUND/AIMS: To investigate the possible role of HIV infection in the natural history of chronic parenterally-acquired hepatitis C. METHODS: A multicenter cross-sectional study was performed in 547 patients with chronic parenterally-acquired hepatitis C with or without HIV infection (116 HIV-positive and 431 HIV-negative). Approximate duration of HCV infection was estimated in all patients included, and histologic diagnoses made at different time intervals following HCV infection were analyzed in both groups. Factors related to serum HCV-RNA levels were also investigated. RESULTS: Histologic findings were similar in liver biopsies from both HIV-infected and noninfected patients. However, in the first 10 years, 13 out of 87 (14.9%) HIV-positive subjects developed cirrhosis, in comparison with 7 out of 272 (2.6%) in the HIV-negative group (p < 0.01). Similar results were found in the first 5 and 15 years, respectively, and most of the HIV-negative patients with cirrhosis (42 out of 56) developed cirrhosis in a time interval longer than 15 years. Consequently, mean interval from estimated time of HCV infection to cirrhosis was significantly longer in HIV-negative than HIV-positive patients (23.2 vs. 6.9 years; p < 0.001). Chronic active hepatitis (with and without cirrhosis) and long duration of HCV infection were significantly associated with higher HCV load (p < 0.05). Finally, HIV-positive patients with CD4+ cell counts > 500 cells/ml showed a lower HCV load than those with < 500 cells/ml (p < 0.05). CONCLUSIONS: HIV infection modifies the natural history of chronic parenterally-acquired hepatitis C with an unusually rapid progression to cirrhosis. HIV-related immunodeficiency may be a determinant of higher hepatitis C viremia levels and more severe liver damage.

Somatostatin plays a dual, stimulatory/inhibitory role in the control of growth hormone secretion by two somatotrope subpopulations from porcine pituitary.

Previous results from our laboratory demonstrated the existence of two subpopulations of porcine somatotropes of low- (LD) and high density (HD) that exhibit differences in ultrastructure and respond in an opposite manner to somatostatin (SRIF) in vitro. In LD cells, SRIF did not affect basal growth hormone (GH) release but partially blocked the stimulatory effect induced by GH-releasing factor (GRF). Conversely, SRIF paradoxically stimulated the secretory activity of HD somatotropes. Here, we have analysed in detail the basic parameters that characterize this differential response. To this end, the time- and dose-dependent effects of SRIF-14 were evaluated on separate monolayer cultures of both subpopulations. Likewise, the direct effect of the peptide on individual somatotropes from each subset was assessed by cell immunoblot assay. Finally, we compared the effects of SRIF-14 and SRIF-28 on cultures of LD and HD cells. SRIF-14 (10(-7) M) induced a rapid (30 min) and sustained (4 h) 2-fold increase in GH release from HD cells, whereas it did not affect GH secretion from LD somatotropes. Surprisingly, a low dose of SRIF (10(-15) M) stimulated GH release from both LD (154.1 +/- 8.2% of basal, P < 0.05) and HD (337.2 +/- 55.5% of basal, P < 0.05) subpopulations, even more effectively than higher doses of the peptide. Results from cell blotting showed that SRIF stimulatory effects were exerted directly upon individual somatotropes. Finally, SRIF-28 elicited similar responses to those observed for SRIF-14 in both somatotrope subpopulations, yet 10(-15) M SRIF-28 was less potent than the same dose of SRIF-14 in stimulating GH release from HD cells. Our present findings demonstrate that SRIF can function as a true GH-releasing factor in cultures of porcine pituitary cells by acting specifically and directly upon somatotropes. Furthermore, together with previous observations, these results strongly suggest that SRIF is not merely an inhibitor of GH release in pigs, but might play a dual modulatory role. Heterogeneity of the somatotrope population contributes greatly to this divergent effect of SRIF.

Different effects of somatostatin on in vitro growth hormone release in two porcine breeds with different growth rates.

A perifusion system of anterior pituitary (AP) tissue was used to investigate the temporal interaction of growth hormone-releasing factor (GRF) and somatostatin (SRIF) in the control of GH secretion in two pig breeds, Göttingen Miniature Pig (GMP), a small obese breed, and German Landrace (GLR), a conventional lean breed. AP tissue pieces derived from sexually mature ovariectomized animals were perifused (6 replicates per treatment) and fractions were collected at 10 min intervals. Basal GH release (ng.ml-1.mg-1 AP) in GLR was twice that of GMP (P < 0.001). Exposure to 10 min pulses of 1 nM GRF repeated 3 times at 2 h intervals resulted in rapid stimulatory GH responses (area under the curve) which became attenuated (P < 0.05) over time in GMP but not in GLR. Surprisingly, during and following the exposure of AP tissue from GMP to 10-, 20-, or 40-min pulses of 10 nM SRIF alone, GH release was markedly stimulated (P < 0.05), while AP tissue from GLR only showed a weak rebound GH release after SRIF pulses. With AP tissue from GLR low concentrations (0.1 nM SRIF) amplified GRF-induced GH release, whereas 1 nM or 10 nM SRIF inhibited GRF-induced GH release. However, concomitant exposure of AP tissue from GMP to 0.1, 1 or 10 nM SRIF during a GRF pulse markedly enhanced the GH response (P < 0.05), compared to 1 nM GRF alone, except for 1 nM SRIF which inhibited the GH response to the first GRF pulse. Thus the presence of SRIF, and not only its withdrawal, is an important factor in setting the timing and duration of GH pulses in both breeds. In GLR the concentration of SRIF is more important than the duration and/or type of SRIF pulse. In contrast, in GMP type and/or duration of SRIF pulses seem to be crucial to optimize pulsatile GH release and even determine peak height of GH pulses caused by GRF. These findings indicate clear breed differences in the role of SRIF and in the control of GH release by the interplay of GRF and SRIF. The "paradoxical' effect of SRIF suggests that the role of SRIF is much more complex than that of a mere inhibitor and whose real role could be a modulator either of GH pulse and/or GRF action on GH release.

Heterogeneity of growth hormone (GH)-producing cells in aging male rats: in vitro GH releasing activity of somatotrope subpopulations.

Studies on the age-related decline of growth hormone (GH) release have ignored that the population of GH-producing cells (somatotropes) is heterogeneous. In aging male rats, centrifugation of dispersed pituitary cells in a density gradient yields two somatotrope subpopulations, i.e. low- (LD) and high-density (HD) cells. A previous analysis of ultrastructure and GH mRNA levels has shown that storage and biosynthetic features were inversely related in both subsets. Furthermore, ultrastructural and molecular differences between LD- and HD-cells were retained throughout the rat lifespan, suggesting that the heterogeneity of somatotropes may have a biological meaning. Accordingly, the main objective of the present study was to analyze the functional heterogeneity of the somatotrope population during the aging process in male rats. For this purpose, the response of LD- and HD-somatotropes from 5-, 19-, and 26-month-old male rats was analyzed with an optimized cell immunoblot assay both under basal conditions, and after GH-releasing factor (GRF) and/or somatostatin (SS) treatments. Simultaneous measurements of hormonal release, intracellular GH content, and cell size were performed at the single-somatotrope level. Average values for those parameters were significantly higher in HD- than in corresponding LD-cells, such differences being irrespective of age or treatment. Releasing activity and GH content were significantly reduced with age in both subpopulations. GRF stimulated GH release from LD- and HD-somatotropes, and the GRF responsiveness was similar in both subpopulations and in all ages. On the other hand, SS prevented GRF-stimulated GH release in most cases. At the level of single cells, both releasing activity and cell size showed a significant, linear dependence on intracellular GH content, correlations being irrespective of age, subpopulation, or treatment. Taken together, our results demonstrate that LD- and HD-somatotrope subpopulations display quantitative differences in releasing activity that are essentially retained through aging. This functional heterogeneity is more dependent on the basal GH release of these somatotrope subsets than in their responsiveness to GRF and SS. The present findings suggest that the reduction in secretory activity at the single somatotrope level observed in both subpopulations underlies the age-related decline of pituitary GH release. Finally, a theoretical model of secretory cycle is proposed which might contribute to the understanding of the biological meaning of the somatotrope subpopulations in aging male rats.

Somatostatin increases growth hormone (GH) secretion in a subpopulation of porcine somatotropes: evidence for functional and morphological heterogeneity among porcine GH-producing cells.

Previous results demonstrate that porcine somatotropes can be separated by density gradient centrifugation into low density (LD) and high density (HD) subpopulations. In rat, two analog somatotrope subpopulations differ morphologically and functionally. In an attempt to determine whether morphological differences were also present within LD and HD porcine somatotropes, we undertook a quantitative electron microscope study of the subcellular organelles of immunoidentified LD and HD somatotropes. In addition, to test for the existence of functional differences, cultures of separated HD and LD subpopulations were treated for 4 h with or without 10 microM GRF-(1-29) and/or 100 microM somatostatin (SRIF), and porcine GH release and intracellular content were evaluated using a homologous enzyme immunoassay. Morphometric results demonstrate that LD somatotropes are smaller in size (P < 0.05) and contain fewer secretory granules (P < 0.05) and more rough endoplasmic reticulum (P < 0.05) than HD somatotropes. In terms of secretion, LD somatotropes showed a classical response; GRF increased GH release 1.7-fold (n = 6; P < 0.05) over the control value, whereas treatment with SRIF alone did not affect basal GH release in this subpopulation, but partially blocked GRF-induced GH release. HD somatotropes responded to GRF with a similar 1.7-fold increase in GH release. However, SRIF administered alone or in combination with GRF exerted a paradoxical stimulatory effect on HD somatotropes (2.15- and 2.12-fold over control value, respectively; n = 6; P < 0.05). These results demonstrate that the porcine somatotrope population is composed of two major subpopulations that display a distinctive pattern of ultrastructural organization and a markedly divergent secretory response to in vitro SRIF treatment.

Heterogeneous response of porcine gonadotrope subpopulations to gonadotropin releasing hormone (GnRH) during postnatal development.

In previous papers, we showed the porcine gonadotrope population to be composed of three GtH subpopulations that can be separated by density using a continuous Percoll density gradient. We also demonstrated that these subpopulations exhibited different hormonal storage patterns and morphological features during porcine postnatal development at three representative ages: neonates (30 days), prepubers (5-6 months) and matures (16-18 months). In this work, we investigated whether these morphologically heterogeneous subpopulations are also functionally different. Thus, the effect of the hypothalamic gonadotropic hormone-releasing factor (GnRH) on these subpopulations was assessed in order to ascertain whether a mutual relationship between the reported morphological features, hormonal storage patterns and physiological response to the stimulation can be established. For this purpose, gonadotropin secretion was measured by cell immunoblot assay and hormonal content by scanning cytophotometry. Low-density gonadotropes (1.049 g/cm3), present in the three age groups studied, were mainly composed of bihormonal LH/FSH cells in neonates and monohormonal LH cells in prepubers and matures. GnRH stimulation was found to increase both LH and FSH secretion, as well as the intracellular content. These results indicate that GnRH can stimulate both the synthesis and release of both gonadotropins in this subpopulation. Middle-density gonadotropes (1.062 g/cm3), present in prepubers and matures only, were composed of bihormonal cells. GnRH stimulated the secretion of LH and FSH in prepubers and matures, but decreased hormonal contents except that of LH in prepubers. However, GnRH stimulation increased the proportion of immunoreactive gonadotropes (particularly monohormonal cells). Finally, high-density cells (1.087 g/cm3), present in neonates and prepubers only, were mostly composed of bihormonal LH/FSH gonadotropes, and exhibited low (neonates) or no response (prepubers) in terms of LH release and content when treated with GnRH. In conclusion, these results indicate that porcine gonadotrope subpopulations are morphologically and physiologically heterogeneous. The heterogeneity remained through porcine postnatal development, thus suggesting that all the subpopulations are physiologically relevant. However, the different hormonal storage patterns between subsets of the same density suggest age-related differences within each subpopulation due, at least in part, to the different physiological condition of the animals during development.