Tratamiento etiológico de trypanosoma cruzi con benznidazol y nifurtimox en áreas rurales / Trypanosoma cruzi etiologic treatment with benznidazole and nifurtimox in rural areas

INTRODUCCIÓN El nifurtimox (NF) y el benznidazol (BZ) se emplean para el tratamiento (TTO) de Trypanosoma cruzi y provocan una caída de la carga parasitaria, que prevendría el desarrollo y/o progresión de la enfermedad. Es importante identificar estrategias que aseguren el acceso temprano al diagnóstico y al TTO, así como una logística para el manejo de efectos adversos (EA). OBJETIVOS Comparar el manejo del TTO con BZ y NF en poblaciones rurales vulnerables. MÉTODOS Se realizó el TTO con BZ a 90 residentes de un área rural de Pampa del Indio en 2010. En 2013, se inició el TTO con NF a 37 pacientes. Las actividades de diagnóstico y TTO fueron planificadas participativamente con la comunidad. Se compararon planificaciones y resultados del TTO (tipo y manejo de EA, adherencia, porcentaje de culminación) entre ambas áreas. La eficacia del TTO se evaluó mediante marcadores moleculares (PCR cuali y cuantitativa). Resultados Completaron el TTO el 85% y el 92% de los pacientes tratados con BZ y NF, respectivamente. El exantema fue el EA más frecuente (28/34) al realizar TTO con BZ, mientras que las cefaleas lo fueron en el TTO con NF (12/20). El tiempo medio de aparición de EA fue de 12 días (BZ) y 11 días (NF). Los pacientes con EA no modificaron el TTO (BZ 53%; NF 80%), lo disminuyeron transitoriamente (BZ 12%; NF 5%) o lo interrumpieron por indicación médica (BZ: 15%; NF 10%) o sin ella (BZ 20%; NF 5%).

Seasonal closures as a measure of trawling effort control in two Mediterranean trawling grounds: effects on epibenthic communities.

Within the framework of ecosystem-based management, we focused on the use of seasonal closures as effective measures to minimise the degradation of benthic communities by trawling. These closures imply the complete cessation of trawling fleet activity and are commonly used in the Mediterranean to reduce the annual fishing effort, with the ultimate goal of effective resource management. In this study, we aimed to investigate how epibenthic communities respond to seasonal closures. The potential benefits of short-term annual closures in two Mediterranean fishing grounds were evaluated by analysing changes in community structure and composition that were linked to the closure. A decrease of faunal abundance was observed with the resumption of fishing activity after the closure at both fishing grounds. Remarkably, results indicated that some large and mobile fauna were able to respond to these closures. We concluded that the currently planned closures are too short to benefit benthic communities.

Trans-sialidase inhibition assay for the detection of Trypanosoma cruzi infection in blood donor samples from Argentina.

BACKGROUND AND OBJECTIVES: Conventional serology tests for Trypanosoma cruzi blood banks screening are neither sensitive nor specific enough, and currently no gold standard assay is available. Trans-sialidase inhibition assay (TIA) detects neutralizing antibodies against T. cruzi trans-sialidase. Conventional serology inconclusive, positive and negative blood donor samples were evaluated by employing TIA as a supplementary test. MATERIALS AND METHODS: Three hundred and twenty-one blood donor samples were tested using a combination of assays. Based on the results of testing, these were divided into a number of groups. All samples were tested by TIA. RESULTS: In conventional serology inconclusive samples 48.1% were TIA-positive, 1/54 conventional serology positive samples was TIA-negative. All negative samples from donors without epidemiological risks were TIA-negative; 1/48 was positive in those with epidemiological risk. CONCLUSION: Trans-sialidase inhibition assay application in blood banks may be useful to resolve inconclusive samples, and thus improves donor counseling and allows individual re-entry. The use of TIA in samples from negative conventional test donors but positive epidemiological antecedents may contribute to decrease transfusional risk.

Regulation of intracellular chloride concentration in rat lactotrope cells and its relation to the membrane resting potential.

Rat lactotrope cells in primary culture exhibit physiological properties closely associated with chloride ions (Cl-) homeostasis. In this work, we studied the regulation of intracellular Cl- concentrations ([Cl-]i) and its relation to the membrane resting potential, using a combination of electrophysiology and spectrofluorimetry. Variations in [Cl-]i resulting from the patch clamp technique, pHi, antagonists of Cl(-)-Ca(2+)-dependent channels, an anion exchanger antagonist, and an antagonist of K(+)-Cl- cotransport were considered with respect to their involvement in membrane potential. We show that: (i) The patch-pipette does not always impose its Cl- concentration. (ii) In rat lactotrope cells, membrane resting potential is partially determined by [Cl-]i. (iii) Besides ion channel activity, electroneutral ion transports (cotransports such as K(+)-Cl- and Na(+)-K(+)-2Cl-) participate actively in maintaining a high [Cl-]i. (iv) Finally, Cl- homeostasis is probably linked to cell energetics.

Interactions between intracellular chloride concentrations, intracellular pH and energetic status in rat lactotrope cells in primary culture.

Rat lactotrope cells in primary cultures have a higher intracellular Cl- concentration ([Cl-]i) than that predicted by a passive distribution across the membrane. This suggests that active cellular mechanisms ensure this ionic equilibrium. In this study, we examined the interactions between pHi, [Cl-]i regulation and cell energetics. We analyzed: 1. the interactions between extracellular Cl- concentrations, [Cl-]i and cellular energy; 2. the influence of [Cl-]i on respiratory chain function; 3. the correlation with glycolysis and; 4. the role played by pHi in these cellular mechanisms. We show that low [Cl-]i decreases ATP cell content, ATP/ADP ratio and modify phosphorylative oxidations. ATP production is rather due to the anaerobic pathway of the glucose metabolism than the aerobic one and depends also on other metabolic substrates among which glutamine probably has a special role. Finally, pHi appears as a determinant in the balance between aerobic and anaerobic pathways. These results are discussed in relation to the role of Cl- in normal and pathological (effect of hypoxia on mature and immature neurons) cell situations.

Effects of ELF and static magnetic fields on calcium oscillations in islets of Langerhans.

Several experimental studies have produced contradictory results on the effects of extremely low frequency (ELF) magnetic fields on cellular processes involving calcium ions. Furthermore, the few positive results have not been independently replicated. In most of these studies, isolated cells were used. Our study used mouse islets of Langerhans, in which very regular oscillations of calcium concentration can be observed at length. These oscillations are sustained by processes that imply energetic and inter-intracellular communication. Various magnetic fields were applied, either sinusoidal at different frequencies (50 Hz or multiples of the natural oscillation frequency) at 0.1 or 1 mT or static at 1 mT. Islets were also exposed to "cyclotron resonance" conditions. There was neither alteration of the fundamental oscillation frequency nor the degree of organisation under all exposure conditions. Using this sensitive model, we could not show new evidence of alterations of calcium processes under exposure to various magnetic fields.

Calcium store depletion induced by mitochondrial uncoupling in prostatic cells.

The effects of mitochondrial uncoupling on the calcium homeostasis of prostatic cells were investigated using the prostatic cancer cell line LNCaP and indo-1 spectrofluorimetry. Carbonyl cyanide m-chloro-phenylhydrazone (CCCP) was used as uncoupler. Resting LNCaP cells responded to CCCP by a biphasic increase in [Ca2+]i. The first phase of increase which corresponded to the release of a mitochondrial CCCP-sensitive Ca2+ store was followed by a second increase phase consisting of Ca2+ influx through the plasma membrane. The relationship between the CCCP- and the InsP3-sensitive stores was investigated using thapsigargin (TG). The release part of the Ca2+ response to TG was reduced in a time-dependent manner by previous exposure of the cells to CCCP, suggesting that CCCP also acts on non-mitochondrial stores. Our results show that CCCP releases Ca2+ from both mitochondrial and non-mitochondrial stores in prostatic cells. The possible mechanisms of these effects are discussed.

Effects of Cl(-) substitution on electrophysiological properties, Ca(2+) influx and prolactin secretion of rat lactotropes in vitro.

In this study, we compared the effects of different chloride (Cl(-)) substitutes - methane sulfonate (CH(3)SO(-)(3)), bromide (Br(-)), nitrate (NO(-)(3)), thiocyanate (SCN(-)) and perchlorate (ClO(-)(4)) - on the secretory activity and calcium current activation of rat lactotropes in primary culture. We observed that CH(3)SO(-)(3) decreased basal prolactin (PRL) secretion. Br(-) had no effect, whereas the more lyotropic anions, such as NO(-3), SCN(-) and C1O(-4), increased basal PRL secretion. The latter three substitutes induced a significant shift in the voltage dependence of T-type calcium channel activation towards hyperpolarized values. However, this shift alone cannot explain the increase in secretion. Anion permeability studies also demonstrated that the organic anion CH(3)SO(-3) was less permeant than Cl(-), whereas monovalent inorganic anions were more permeant, with the following anion permeability sequence: SCN(-) > ClO(-4) > NO(-3) > Br(-). In conclusion, deprivation of Cl(-) ions has converse consequences on basal and induced secretion; permeating anions result in a transient increase in intracellular Ca(2+) ions. This process involves voltage-dependent Ca(2+) channels. We propose that an alteration in intracellular anion concentrations may influence the activation of internal effectors such as G proteins or channel proteins and, therefore, interfere with exocytosis. These effects are correlated with an external action of lyotropic anions, particularly NO(-3), ClO(-4) and SCN(-), on the gating properties of T-type calcium channels, probably through changes in cell surface charges. The results demonstrate the modulatory effect of anions on the secretory activity of rat lactotropes and underline the specific role played by chloride in stimulus-secretion coupling.

Regulation of intracellular pH in rat lactotrophs: involvement of anionic exchangers.

Regulation of the intracellular pH (pHi) of normal rat lactotrophs was studied. As this cell type, cultured with 10% FCS, can achieve a relatively alkaline pHi (7.3-7.5), we investigated the presence of a mechanism based on Cl-/HCO3- exchange. Using the pHi-sensitive probe SNARF-1 (seminaphtorodafluor) in its permeant form, SNARF-1/AM, we studied pHi recovery after acidic loading in individual cells with a microspectrofluorometric approach. We showed the involvement of anionic exchange in lactotroph cell pHi regulation. Acute CO2-bicarbonate cell acidic loading combined with external Cl- depletion induces the activation of a Cl-/HCO3- exchange. This exchange is 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid sensitive and corresponds to the type 3 anionic exchanger (AE3). However, after nigericin acidification, Na+/H+ exchange can also participate in recovery. In addition, incubation experiments strongly suggest that a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-insensitive anionic exchanger (type 2 anionic exchanger or AE2) is present in rat lactotrophs. The presence and involvement of carbonic anhydrase in pHi regulation have been demonstrated. Finally, using Northern blot and reverse transcription-PCR techniques, messenger RNAs for both AE2 and AE3 were identified in anterior pituitary cell extracts. We concluded that in normal rat lactotrophs, pHi regulation is achieved by a complex system in which Cl-/HCO3- exchange has a pivotal role.

An mRNA encoding a putative GABA-gated chloride channel is expressed in the human cardiac conduction system.

GABA-gated chloride channels are the main inhibitory neurotransmitter receptors in the CNS. Conserved domains among members of previously described GABAA receptor subunits were used to design degenerate sense and antisense oligonucleotides. A PCR product from this amplification was used to isolate a full-length cDNA. The predicted protein has many of the features shared by other members of the ligand-gated ion channel family. This channel subunit has significant amino acid identity (25-40%) with members of GABAA and GABAC receptor subunits and thus may represent a new subfamily of the GABA receptor channel. Although we cannot rule out that this clone encodes a receptor for an unidentified ligand, it was termed GABA chi. This gene is mainly expressed in placenta and in heart; however, placenta appears to express only an unspliced mRNA. In situ hybridization reveals that the GABA chi subunit mRNA is present in the electrical conduction system of the human heart. Our results suggest that novel GABA receptors expressed outside of the CNS may regulate cardiac function.

Modulation of voltage-dependent Ca2+ conductance by changing Cl- concentration in rat lactotrophs.

In pituitary cells, voltage-dependent Ca2+ channels play an important role in such physiological processes as exocytosis, secretion, the cell cycle, and proliferation. Thus mechanisms that modulate voltage-dependent Ca2+ channel activity participate indirectly in regulating intracellular Ca2+ concentration. We have shown a new modulating mechanism for voltage-dependent Ca2+ channels by demonstrating that Ca2+ influx is influenced by Cl-. To evaluate the role of Cl- on Ca2+ conductance coupling, we first measured the intracellular Cl- concentration of rat lactotrophs using the Cl(-)-sensitive fluorescence probe sulfopropylquinolinium by simple microspectrofluorometry or combined with electrophysiology. We found an average intracellular Cl- concentration of rat lactotrophs of approximately 60 mM (n = 39). Using the whole cell tight-seal recording technique, we showed that a reduction in external Cl- concentration ([Cl-]o) and a decrease in Cl- conductances affected Ca2+ conductance as measured by Ba2+ movement through the Ca2+ channels (I(Ba)). Low [Cl-]o (39 mM) induced a decrease in Ca2+ entry via voltage-gated Ca2+ channels (-27.75 +/- 4% of normalized I(Ba)). Similarly, blockade of the Cl- conductance by 1 mM 9-anthracene carboxylic acid induced a decrease in I(Ba) (-26 +/- 6% of normalized I(Ba)). This modulation of I(Ba) was inhibited by 24-h pretreatment of the cells with pertussis toxin (1 microg/ml), suggesting that changes in Cl- concentration induced by low [Cl-]o and 9-anthracene carboxylic acid interfered with the phosphorylation of G proteins involved in Ca2+ channel activation. These results suggest a feedback mechanism based on constant interaction between Ca2+ and Cl-. Finally, they also emphasize the physiological role of Cl- in rat lactotrophs.

Regulation of intracellular chloride concentration in rat lactotrophs: possible role of mitochondria.

Increasing evidence is accumulating for the involvement of chloride ions in the stimulus-secretion coupling of pituitary cells. We show that the mean intracellular chloride concentration [Cl-]i of rat lactotroph cells maintained in culture is high, close to 60 mM (59.4 mM), using the Cl- sensitive fluorescent probe 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ), coupled with whole-cell patch-clamp recordings. We demonstrate that this high level is correlated with the presence of mitochondrial stores of Cl- as shown by the release of Cl- in response to various metabolic inhibitors. We determine that CCP (50 microM) induces a mean [Cl-]i increase of 15.8+/-5.8 mM, using combined electrophysiology and microspectrofluorimetry methods. These data strongly suggest that cell metabolism, including the mitochondrial function, modulate [Cl-]i.

Recording of a large-conductance chloride channel in normal rat lactotrophs.

Membrane current fluctuations resembling channel openings and closings were observed in the whole cell configuration of the patch-clamp technique in normal rat lactotrophs in primary culture. Using high-gain head stage in whole cell configuration, we characterized the nature and pharmacological properties of the ionic channel underlying these fluctuations. This channel, found in small numbers (< 10 per cell), was specific for Cl- because its reversal potential varied with Cl- gradients, according to the Nernst equation, and its unitary amplitude was linearly related to membrane potential from -100 to 0 mV. Slope conductance was close to 100 pS. Analyzing open times, we demonstrated its Ca2+ and potential dependence. Four sublevels were observed. We suggest that this channel, belonging to the background Cl- channel group, takes part in the regulation of intracellular Cl- concentration of normal rat lactotrophs.

Long-term effects of calcium availability on prolactin and protein synthesis in human decidual cells.

Ever since decidual cells were recognized as the source of decidual prolactin (dPRL), very few reports have dealt with the role of calcium (Ca2+) on dPRL synthesis and release. In a recent work, we described the presence of T-type Ca2+ channels in these cells, giving Ca(2+)-dependent action potentials. However, we failed to demonstrate any action of decidual cell Ca2+ modulation on acute dPRL release, but observed only long-term effects. We have now investigated these effects on decidual protein and dPRL synthesis after 24 h treatments. When Ca2+ channel blockers or EGTA (2 mM) were added to the culture medium, dPRL release and [3H] leucine incorporation into proteins decreased. Increasing external Ca2+ up to 2 mM instead of 0.8 mM or changing the external K+ concentration (30 mM instead of 5.6) had no consequence on dPRL release, whereas 2 mM of Ca2+ enhanced total protein synthesis. No toxicity was noted with these treatments. Finally a possible effect of Ca2+ modulation on dPRL synthesis was studied using [35S] methionine. The specific activity of [35S] methionine on dPRL was similar in control and treated cells (EGTA, 2 mM Ca2+, cobalt). These results support the idea that Ca2+ controls dPRL synthesis in decidual cells, acting only on general protein synthesis processes.

Biphasic changes in intracellular pH induced by thyrotropin-releasing hormone in pituitary cells.

We studied the effects of TRH on intracellular pH (pHi) in individual cells of the GH3 pituitary clonal cell line using the seminaphtorhodafluor pH indicator. We show that, in a majority of cells, TRH action on pHi occurs in two phases: first acidification then alkalinization. Acidification and Ca2+ mobilization are related in time. K+ depolarization (KCl, 50 mM), and Ca2+ ionophores, A23187 (10 microM) or ionomycin (5 microM) lead to acidification. We conclude that a marked increase in [Ca2+]i can induce acidification and that the TRH-induced acidification is due to Ca2+ mobilization. TRH-induced alkalinization is due to Na+/H+ exchanger activation, since it is inhibited by amiloride (200 microM) and Na(+)-free medium. We show that this alkalinization does not occur after a 20-h pretreatment with phorbol myristate acetate (1 microM) which depletes protein kinase C. We also show that blocking Ca2+ entry does not affect the TRH-induced alkalinization, but an increase in [Ca2+]i concomitant with the activation of protein kinase C mimics TRH-induced alkalinization. We conclude that both Ca2+ mobilization and protein kinase C activation are necessary for TRH-induced alkalinization. Studies of secretion in Na(+)-free medium or with amiloride (200 microM) show that pHi does not seem to be involved in PRL short-term release (30 min) but suggest that activation of the Na+/H+ exchanger leading to cytoplasmic alkalinization may have an important role in PRL synthesis.

Gonadotropin-releasing hormone-induced changes of intracellular pH in pituitary gonadotrophs: influence of estradiol.

Using the pH indicator, seminaphtorhodafluor, we studied the effects of GnRH on intracellular pH (pHi) in single gonadotroph cells, obtained from 3-week ovariectomized rats, treated or not with estradiol (E2) (OVX + E2, OVX). In a majority of cells (77.7% for OVX cells and 93.7% for OVX + E2 cells), GnRH induced acidification. A biphasic change of pHi, acidification followed by alkalinization, was observed in about 44% of the cells tested. In E2-treated cells, amplitude of acidification and duration of alkalinization were increased. Acidification and Ca2+ mobilization were related in time with a short delay (4-5 sec.). Depolarization with KCl and ionomycin, a Ca2+ ionophore, induced acidification. Taken together these observations suggest that acidification was caused by [Ca2+]i increase. When the Na+/H+ exchanger was blocked by amiloride or in Na(+)-free medium, GnRH-induced alkalinization was inhibited. Alkalinization disappeared completely when the cells were depleted in protein kinase C (PKC). Nevertheless, acute application of phorbol myristate acetate, known to activate PKC, was not sufficient to induce alkalinization. We conclude that PKC is necessary but not sufficient for alkalinization. In contrast, the GnRH response can be mimicked by a simultaneous application of phorbol myristate acetate and KCl. To further explore the putative role of pHi in the secretory process, LH release was studied. Using Na(+)-free medium or amiloride, we show that basal LH was not dependent upon the Na+/H+ exchanger activity. Conversely, GnRH-induced LH release was significantly decreased; this decrease was greater in E2-treated cells but prevented by bicarbonate. These data show that pHi and the Na+/H+ exchanger play an important role in the stimulus secretion coupling process of gonadotrophs. E2, which is an important factor in the regulation of gonadotropic hormone release, participates also in the pHi variations.

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.

Voltage-dependent calcium current in human decidual cells and its relation to prolactin secretion.

Human decidual cells synthesize and release decidual PRL (dPRL) immunologically and biochemically identical to human pituitary PRL. However, stimulators and inhibitors of PRL secretion such as TRH, bromocriptine or dopamine have no effect on dPRL release. The evidence for the involvement of Ca2+ in dPRL release is based on contradictory or unclear data. Since little is known about Ca2+ movement in human decidual cells we studied the membrane Ca2+ conductance of cultured decidual cells using the patch-clamp technique in the whole-cell configuration. We report the existence of Ca(2+)-dependent action potentials triggered by hyperpolarizing or depolarizing pulses and blocked by cobalt (Co2+; 5 mM). Spontaneous action potentials were observed in the cell-attached mode and found also to be Co(2+)-sensitive. A tetrodotoxin-insensitive and Ca(2+)-dependent rapidly inactivating inward current was investigated in voltage clamp. Its activation threshold was between -60 and -45 mV. Indo-1 measurements of free intracellular Ca2+ concentrations ([Ca2+]i, 169 +/- 14 nM and 141 +/- 8 nM in short-term culture vs. 149 +/- 5 nM in cells cultured for 3-6 days) showed that decidual cells have spontaneous transient fluctuations of [Ca2+]i and that [Ca2+]i was decreased by Ca2+ channel blockers. The existence of Ca2+ movements in decidual cells in culture is thus demonstrated. The occurrence of action potentials in decidual cells derived from fibroblasts, reputed to be inexitable cells, is an interesting biological observation. However, Ca2+ is not involved in the short-term release of PRL by decidual cells, and its effects on long-term secretion still requires further investigation.

Calcium-activated chloride conductance of lactotrophs: comparison of activation in normal and tumoral cells during thyrotropin-releasing-hormone stimulation.

We studied a chloride (Cl-) conductance activated by calcium (Ca2+) in normal rat lactotrophs and compared its activation during TRH stimulation in normal rat lactotrophs and in GH3 tumoral lactosomatotrophs cells, using the whole-cell configuration of the patch-clamp technique. The Cl- specificity of the conductance was assessed by manipulation of internal and external Cl- concentrations. The reversal potentials were in agreement with those predicted by the Nernst equation. Ca2+ ionophore A23187 and membrane depolarizations activated the Cl- conductance. However, a feedback effect of Cl- gradient modifications on Ca2+ movements was also observed in normal lactotrophs. In the latter, TRH (100 nM) mobilization of intracellular Ca2+ activated this Cl- conductance together with the potassium (K+) conductance when both ions were present in the intracellular medium (IM) or alone when K+ was absent. Chloride conductance was not activated in the GH3 cells, where mobilization of intracellular Ca2+ by TRH (100 nM) activated only Ca2(+)-dependent K+ conductance. It seems likely that the activation of Cl- conductance in these two different cell types involves different mechanisms.

Growth hormone-releasing factor stimulates calcium entry in the GH3 pituitary cell line.

The GH3 pituitary cell line has been extensively used to study various aspects of the stimulus secretion coupling process. It is known that GH3 cells release PRL and GH in the basal state and in response to various secretagogues. However, this cell line was considered unsuitable as a model for studying the effects of GHRF since the neuropeptide did not affect GH secretion or gene expression. This suggested that the GH3 cells may lack GHRF receptors. The present study investigates the effect of GHRF on free intracellular Ca2+ concentrations in GH3 cells. Cytosolic free calcium concentrations ([Ca2+]i) were monitored in individual cells by microspectrofluorimetry using the fluorescent dye indo 1. When the cells were challenged with a brief application of GHRF (100 nM; 15 sec), 36 out of 59 of these cells responded within a few seconds by a marked increase in [Ca2+]i. GHRF enhanced the frequency of [Ca2+]i oscillations in spontaneously active cells or triggered [Ca2+]i oscillations in inactive cells. The response to GHRF was totally blocked by external Ca2+ free solutions and Ca2+ channel blockers. Combined electrophysiological and fluorescent experiments were carried out in 16 cells. Eleven responded to GHRF. In all cases, the Ca2+ transients triggered by GHRF were associated with action potentials. The Ca2+ responses observed in our experiments clearly show that GH3 cells possess membrane receptors to GHRF. Thus, it is likely that the lack of secretory response observed in GH3 cells does not result from the absence of binding sites to the peptide. It is more likely to be related to alterations of transduction mechanisms resulting in uncoupling between stimulation and secretion.