Low-threshold exocytosis induced by cAMP-recruited CaV3.2 (alpha1H) channels in rat chromaffin cells.

We have studied the functional role of CaV3 channels in triggering fast exocytosis in rat chromaffin cells (RCCs). CaV3 T-type channels were selectively recruited by chronic exposures to cAMP (3 days) via an exchange protein directly activated by cAMP (Epac)-mediated pathway. Here we show that cAMP-treated cells had increased secretory responses, which could be evoked even at very low depolarizations (-50, -40 mV). Potentiation of exocytosis in cAMP-treated cells did not occur in the presence of 50 microM Ni2+, which selectively blocks T-type currents in RCCs. This suggests that the "low-threshold exocytosis" induced by cAMP is due to increased Ca2+ influx through cAMP-recruited T-type channels, rather than to an enhanced secretion downstream of Ca2+ entry, as previously reported for short-term cAMP treatments (20 min). Newly recruited T-type channels increase the fast secretory response at low voltages without altering the size of the immediately releasable pool. They also preserve the Ca2+ dependence of exocytosis, the initial speed of vesicle depletion, and the mean quantal size of single secretory events. All this indicates that cAMP-recruited CaV3 channels enhance the secretory activity of RCCs at low voltages by coupling to the secretory apparatus with a Ca2+ efficacy similar to that of already existing high-threshold Ca2+ channels. Finally, using RT-PCRs we found that the fast inactivating low-threshold Ca2+ current component recruited by cAMP is selectively associated to the alpha1H (CaV3.2) channel isoform.

Exposure to cAMP and beta-adrenergic stimulation recruits Ca(V)3 T-type channels in rat chromaffin cells through Epac cAMP-receptor proteins.

T-type channels are expressed weakly or not at all in adult rat chromaffin cells (RCCs) and there is contrasting evidence as to whether they play a functional role in catecholamine secretion. Here we show that 3-5 days after application of pCPT-cAMP, most RCCs grown in serum-free medium expressed a high density of low-voltage-activated T-type channels without altering the expression and characteristics of high-voltage-activated channels. The density of cAMP-recruited T-type channels increased with time and displayed the typical biophysical and pharmacological properties of low-voltage-activated Ca(2+) channels: (1) steep voltage-dependent activation from -50 mV in 10 mm Ca(2+), (2) slow deactivation but fast and complete inactivation, (3) full inactivation following short conditioning prepulses to -30 mV, (4) effective block of Ca(2+) influx with 50 microM Ni(2+), (5) comparable permeability to Ca(2+) and Ba(2+), and (6) insensitivity to common Ca(2+) channel antagonists. The action of exogenous pCPT-cAMP (200 microM) was prevented by the protein synthesis inhibitor anisomycin and mimicked in most cells by exposure to forskolin and 1-methyl-3-isobutylxanthine (IBMX) or isoprenaline. The protein kinase A (PKA) inhibitor H89 (0.3 microM) and the competitive antagonist of cAMP binding to PKA, Rp-cAMPS, had weak or no effect on the action of pCPT-cAMP. In line with this, the selective Epac agonist 8CPT-2Me-cAMP nicely mimicked the action of pCPT-cAMP and isoprenaline, suggesting the existence of a dominant Epac-dependent recruitment of T-type channels in RCCs that may originate from the activation of beta-adrenoceptors. Stimulation of beta-adrenoceptors occurs autocrinally in RCCs and thus, the neosynthesis of low-voltage-activated channels may represent a new form of 'chromaffin cell plasticity', which contributes, by lowering the threshold of action potential firing, to increasing cell excitability and secretory activity during sustained sympathetic stimulation and/or increased catecholamine circulation.

BDNF up-regulates evoked GABAergic transmission in developing hippocampus by potentiating presynaptic N- and P/Q-type Ca2+ channels signalling.

Chronic application of brain-derived neurotrophic factor (BDNF) induces new selective synthesis of non-L-type Ca2+ channels (N, P/Q, R) at the soma of cultured hippocampal neurons. As N- and P/Q-channels support neurotransmitter release in the hippocampus, this suggests that BDNF-treatment may enhance synaptic transmission by increasing the expression of presynaptic Ca2+ channels as well. To address this issue we studied the long-term effects of BDNF on miniature and stimulus-evoked GABAergic transmission in rat embryo hippocampal neurons. We found that BDNF increased the frequency of miniature currents (mIPSCs) by approximately 40%, with little effects on their amplitude. BDNF nearly doubled the size of evoked postsynaptic currents (eIPSCs) with a marked increase of paired-pulse depression, which is indicative of a major increase in presynaptic activity. The potentiation of eIPSCs was more relevant during the first two weeks in culture, when GABAergic transmission is depolarizing. BDNF action was mediated by TrkB-receptors and had no effects on: (i) the amplitude and dose-response of GABA-evoked IPSCs and (ii) the number of GABA(A) receptor clusters and the total functioning synapses, suggesting that the neurotrophin unlikely acted postsynaptically. In line with this, BDNF affected the contribution of voltage-gated Ca2+ channels mediating evoked GABAergic transmission. BDNF drastically increased the fraction of evoked IPSCs supported by N- and P/Q-channels while it decreased the contribution associated with R- and L-types. This selective action resembles the previously observed up-regulatory effects of BDNF on somatic Ca2+ currents in developing hippocampus, suggesting that potentiation of presynaptic N- and P/Q-channel signalling belongs to a manifold mechanism by which BDNF increases the efficiency of stimulus-evoked GABAergic transmission.

Analysis of the FHIT gene and its product in squamous cell carcinomas of the head and neck.

The FHIT gene has been implicated as a tumor suppressor gene in human malignancies. To determine if FHIT alterations play a role in human squamous cell carcinogenesis of the head and neck (HNSCC), we examined the gene and its product by RT-PCR, SSCP, Northern, Southern, and Western blot analysis in primary HNSCC and/or HNSCC cell lines. Three of 32 tumor samples lacked detectable expression of FHIT by RT-PCR but showed amplification of a control gene of similar size. One of 29 primary tumors and 2/9 HNSCC cell lines exhibited aberrant transcripts generated by RT-PCR methods using one set of 40 cycles of amplification. FHIT mRNA expression was absent in seven HNSCC cell lines but detectable in primary keratinocytes by Northern analysis. Using specific polyclonal antiserum to the full-length FHIT protein in immunoblot analyses, 4/9 cell lines analysed showed no expression of pFhit, two exhibited low levels of expression, and three expressed a putative truncated pFhit. One of 15 tumors analysed also exhibited an overexpressed truncated protein. PCR/SSCP and Southern analysis of one cell line DNA that expressed a truncated protein indicated that it sustained homozygous loss of FHIT exon 5. Our results suggest that alterations in FHIT at the DNA, RNA, and protein levels exist at a low but significant frequency in HNSCCs. Further studies regarding the potential biological activity of FHIT are needed to clarify the role of this gene in HNSCC tumorigenesis.

The Habitability Mini-Laboratory: testing the tools of space habitat architecture.

Living in the closed, confined environment of a space station for a long period and under microgravity conditions, crew members can encounter problems of a physiological and also a psychological nature. The architecture of their living quarters can greatly influence their well-being and their efficiency. Simulation of a proposed architecture and of human movement within that architecture is the most effective way to evaluate the design. Two simulation tools, Computer-Aided Design (CAD) software tools and a mock-up on a smaller scale, were used to 'construct' several proposed architectures. Those models were then evaluated to determine the investigation methods that should be used in future architectural development projects.

European isolation and confinement study. Additional experiments.

Microbiological Experiments. The ISEMSI microbiological contamination experiments confirmed known hypotheses, such as: the trend toward uniformity of skin microbial flora across a group of individuals enclosed together; the rather fast "colonization" of the environment by microorganisms shed by human inhabitants; and the heavy growth of microorganisms in poorly accessible and wet areas (toilets, air conditioning). In addition, possible disturbances of skin defense mechanisms against colonization by potentially pathogenic microbes were noted, as well as a difficulty in monitoring the microbial contents of the atmosphere (significant random variations occur between samples taken at different times and locations). Sensors for Atmospheric Contaminants. Several different prototypes of "array sensors" for the monitoring of trace gas contaminants in the atmosphere were evaluated during ISEMSI. Their performance was promising when compared with results achieved with a more conventional (and more complex) gas chromatograph/mass spectrometer device, also used during ISEMSI. An overall picture of the most important chemical contaminants to be found in enclosed, manned habitats (including contaminants produced by man himself) was obtained via the use of Tenax gas-adsorption traps. This permitted monitoring the fluctuation of contaminants on a daily basis, as well as during the complete 4-week period. Results will provide a valuable input for designing systems to monitor and control atmospheric contamination in future spacecraft. Particular attention was devoted to the monitoring of carbon monoxide in the chamber. Results showing the correlation between its concentration in the atmosphere and the percentage of carboxyhemoglobin in the EMSInauts' blood will allow the evaluation of the correctness of the presently specified maximum allowable concentration for spacecraft. Telemedicine Experiment. The telemedicine experiment confirmed the feasibility and importance of applying to a space station scenario many aspects of remote health care already widely used in the maritime environment. ISEMSI successfully evaluated telemedical consultation procedures and training protocols for the crew. EMSInauts, trained as paramedical assistants, had to interview a "patient" (another EMSInaut, trained to feign illness symptoms), prepare an anamnesis, carry out a medical examination, assess the severity of the case, and administer effective medical care under remote medical advice. An expert system was used to provide step-by-step guidance to the paramedical assistant. ISEMSI demonstrated the great importance of practicing and rehearsing emergency procedures; it confirmed that simulation of medical emergencies during an actual long-duration space mission will be required to provide "refresher training" to astronauts trained as paramedical assistants.(ABSTRACT TRUNCATED AT 400 WORDS)

Global approach to simulation: a gateway to long-term human presence in space.

The establishment of an autonomous European manned space capability is an objective set up by the ESA Council Meeting at the ministerial level, in 1985/1987. ESA's Long-Term Programme Office (LTPO), charged of the preparation of the programme for a European Manned Space Infrastructure (EMSI), started during 1988 to build up an intellectual framework in the domain of long-duration manned space missions. EMSI scope was eventually extended to embrace Moon/Mars missions and bases. Several exploratory studies on problems related to human factors in long-duration space missions were initiated by LTPO. The work of an ad-hoc group of experts (SIMIS Group) has been focused during 1989/1990 on the planning for simulation of such missions with a broad mandate, covering the physiological, psychological and operational aspects of long-duration exposure to microgravity and isolation/confinement. Preliminary results of SIMIS activities are reported. The HYDREMSI experiment, carried out in a terrestrial, analogous environment for 72 days during 1989, is described as an example of the envisaged simulations.