Marcelo Ozu

Serotonin in Golden Hamster Testes: Testicular Levels, Immunolocalization and Role during Sexual Development and Photoperiodic Regression-Recrudescence Transition

Serotonin (5-HT) is found in the gonads and accessory reproductive organs of several species. The golden (Syrian) hamster is a seasonal breeder. Exposure of male adult hamsters to short days for 14 weeks results in a severe gonadal regression, while after a photoinhibition period of 22 weeks a spontaneous testicular recrudescence occurs. The aim of this study was to investigate the presence of 5-HT and its major metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the gonads of golden hamsters, its immunolocation and its physiological role in the testis. The influence of age and photoperiod was also analyzed. Hamsters of 23, 36, 46, 60 and 90 days of age were kept in long photoperiod (LP: 14:10 h light/dark), and adult animals were exposed either to LP or to short photoperiod (SP: 6:18 h light/dark) for 14 and 22 weeks. Testicular parenchyma and capsule levels of 5-HT and 5-HIAA increased significantly at ages of 36 and 60–90 days, but decreased markedly during the exposure of adult hamsters to SP for 14 and 22 weeks. Mast cells were found exclusively in the testicular capsule. The testicular number of mast cells increased concomitantly with age, but decreased in adult hamsters exposed to SP. Mast and Leydig cells presented 5-HT-positive immunoreactivity. During sexual maturation as well as during the transfer of adult hamsters from LP to SP, the 5-HIAA/5-HT ratio showed the highest values in active adult animals, indicating that the increase in testicular 5-HT levels in adulthood is accompanied by an augment in 5-HT turnover. In vitro basal and hCG-stimulated testosterone production was significantly inhibited in presence of physiological concentrations of 5-HT. In conclusion, the present studies demonstrate the existence of 5-HT in mast cells and Leydig cells of hamster testes, as well as describe an inhibitory action of this neurotransmitter on gonadal testosterone production. Furthermore, the age-dependent and photoperiodic-related changes detected in testicular 5-HT levels suggest that this neurotransmitter might act as an important local modulator of the action of gonadotropins on steroidogenesis during sexual development and during the photoperiodic regression-recrudescence transition in the golden hamster.

Human AQP1 Is a Constitutively Open Channel that Closes by a Membrane-Tension-Mediated Mechanism

This work presents experimental results combined with model-dependent predictions regarding the osmotic-permeability regulation of human aquaporin 1 (hAQP1) expressed in Xenopus oocyte membranes. Membrane elastic properties were studied under fully controlled conditions to obtain a function that relates internal volume and pressure. This function was used to design a model in which osmotic permeability could be studied as a pressure-dependent variable. The model states that hAQP1 closes with membrane-tension increments. It is important to emphasize that the only parameter of the model is the initial osmotic permeability coefficient, which was obtained by model-dependent fitting. The model was contrasted with experimental records from emptied-out Xenopus laevis oocytes expressing hAQP1. Simulated results reproduce and predict volume changes in high-water-permeability membranes under hypoosmotic gradients of different magnitude, as well as under consecutive hypo- and hyperosmotic conditions. In all cases, the simulated permeability coefficients are similar to experimental values. Predicted pressure, volume, and permeability changes indicate that hAQP1 water channels can transit from a high-water-permeability state to a closed state. This behavior is reversible and occurs in a cooperative manner among monomers. We conclude that hAQP1 is a constitutively open channel that closes mediated by membrane-tension increments.

Uroguanylin Regulates Net Fluid Secretion via the NHE2 Isoform of the Na+/H+ Exchanger in an Intestinal Cellular Model

Uroguanylin (UGN) has been proposed as a key regulator of salt and water intestinal transport. Uroguanylin activates cell-surface guanylate cyclase C receptor (GC-C) and modulates cellular function via cyclic GMP (cGMP), thus increasing electrolyte and net water secretion. It has been suggested that the action of UGN could involve the Na+/H+ exchanger, but the actual contribution of this transporter still remains unclear. The objective of our study was to investigate the putative effects of UGN on some members of the Na+/H+ exchanger family (NHEs), as well as to clarify its consequences on transepithelial fluid flow in T84 cells. In order to do so, transepithelial fluid flow (Jv) was studied by optic techniques and intracellular pH (pHi) was measured with a fluorescence method. Results showed that NHE2 is found at the apical membrane and has a major role in Na+ absorption; NHE1 and NHE4 are localized at the basolateral membrane with a house-keeping role in steady state pHi. In the assayed conditions, cell exposure to apical UGN increases net secretory Jv, without changing short-circuit currents nor transepithelial resistance, and reduces NHE2 activity. Therefore, at physiological pH, the effect on net Jv was produced mainly by a reduction in normal Na+ absorption through NHE2, rather than by the stimulation of electrolyte secretion. Our study shows that the effect of UGN on pHi is GC-C/cGMP-mediated and enhanced by sildenafil, thus involving PDE5 enzyme. Additionally, cell exposure to apical UGN results in intracellular alkalinization, probably due to indirect effects on basolateral NHE1 and NHE4, which have a major role in pHi regulation.

Simple and inexpensive hardware and software method to measure volume changes in Xenopus oocytes expressing aquaporins.

Water channels (aquaporins) family members have been identified in central nervous system cells. A classic method to measure membrane water permeability and its regulation is to capture and analyse images of Xenopus laevis oocytes expressing them. Laboratories dedicated to the analysis of motion images usually have powerful equipment valued in thousands of dollars. However, some scientists consider that new approaches are needed to reduce costs in scientific labs, especially in developing countries. The objective of this work is to share a very low-cost hardware and software setup based on a well-selected webcam, a hand-made adapter to a microscope and the use of free software to measure membrane water permeability in Xenopus oocytes. One of the main purposes of this setup is to maintain a high level of quality in images obtained at brief intervals (shorter than 70 ms). The presented setup helps to economize without sacrificing image analysis requirements.

Molecular dynamics of water in the neighborhood of aquaporins

Present knowledge obtained by molecular dynamics (MD) simulation studies regarding the dynamics of water, both in the vicinity of biological membranes and within the proteinaceous water channels, also known as aquaporins (AQPs), is reviewed. A brief general summary of the water models most extensively employed in MD simulations (SPC, SPC/E, TIP3P, TIP4P), indicating their most relevant pros and cons, is likewise provided. Structural considerations of water are also discussed, based on different order parameters, which can be extracted from MD simulations as well as from experiments. Secondly, the behaviour of water in the neighbourhood of membranes by means of molecular dynamics simulations is addressed. Consequently, the comparison with previous experimental evidence is pointed out. In living cells, water is transported across the plasma membrane through the lipid bilayer and the aforementioned AQPs, which motivates this review to focus mostly on MD simulation studies of water within AQPs. Relevant contributions explaining peculiar properties of these channels are discussed, such as selectivity and gating. Water models used in these studies are also summarised. Finally, based on the information presented here, further MD studies are encouraged.

Mechanisms of interaction between Candida albicans and Streptococcus mutans: An experimental and mathematical modelling study

Abstract Objective. To evaluate the mechanisms of microbial interaction between the oral pathogens Candida albicans and Streptococcus mutans. Materials and methods. Growth kinetics for the two micro-organisms, cultured individually or together, were followed experimentally for 36 h. The different growth curves were analysed by means of mathematical modelling. Results. Under the experimental conditions, S. mutans final concentration, when grown individually, was 5-times that of C. albicans. Contrarily, when both micro-organisms grew together, this ratio was inversed and C. albicans final concentration was even higher than that of S. mutans. When both micro-organisms share the niche, a model including linear competition among one another was best suited to reproduce the experimental observations. The results of this model show that the initial growth rates of both species are positively influenced by their mutual interaction. However, at longer incubation times, C. albicans prevents bacterial growth and achieves concentrations 4-times higher than when grown individually. Conclusions. The results suggest that C. albicans biofilm formation could be potentiated by the presence of S. mutans by two mechanisms: synergically at short times and by competition at longer periods.

ENaC Channels in Oocytes from Xenopus laevis and their Regulation by xShroom1 Protein

Shroom is a family of related proteins linked to the actin cytoskeleton. xShroom1 is constitutively expressed in X. oocytes and is required for the expression of amiloride sensitive sodium channels (ENaC). Oocytes were injected with α, β, and γ mENaC and xShroom1 sense or antisense oligonucleotides. We used voltage clamp techniques to study the amiloride-sensitive Na+ currents (INa(amil)). We observed a marked reduction in INa(amil) in oocytes co-injected with xShroom1 antisense. Oocytes expressing a DEG mutant β-mENaC subunit (β-S518K) with an open probability of 1 had enhanced INa(amil) although these currents were also reduced when co-injected with xShroom1 antisense. Addition of low concentration (20 ng/ml) of trypsin which activates the membrane-resident ENaC channels led to a slow increase in INa(amil) in oocytes with xShroom1 sense but had no effect on the currents in oocytes coinjected with ENaC and xShroom1 antisense. The same results were obtained with higher concentrations of trypsin (2 µg/ml) exposed during 2.5 min. In addition, fluorescence positive staining of plasma membrane in the oocytes expressing α, β and γ mENaC and xShroom1 sense were observed but not in oocytes coinjected with ENaC and xShroom1 antisense oligonucleotides. On this basis, we suggest that xShroom1-dependent ENaC inhibition may be through the number of channels inserted in the membrane.

A counterpoint between computer simulations and biological experiments to train new members of a laboratory of physiological sciences

When new members join a working group dedicated to scientific research, several changes occur in the groups dynamics. From a teaching point of view, a subsequent challenge is to develop innovative strategies to train new staff members in creative thinking, which is the most complex and abstract skill in the cognitive domain according to Blooms revised taxonomy. In this sense, current technological and digital advances offer new possibilities in the field of education. Computer simulation and biological experiments can be used together as a combined tool for teaching and learning sometimes complex physiological and biophysical concepts. Moreover, creativity can be thought of as a social process that relies on interactions among staff members. In this regard, the acquisition of cognitive abilities coexists with the attainment of other skills from psychomotor and affective domains. Such dynamism in teaching and learning stimulates teamwork and encourages the integration of members of the working group. A practical example, based on the teaching of biophysical subjects such as osmosis, solute transport, and membrane permeability, which are crucial in understanding the physiological concept of homeostasis, is presented.

Tonoplast (BvTIP1;2) and plasma membrane (BvPIP2;1) aquaporins show different mechanosensitive properties

Previous works proposed that aquaporins behave as mechanosensitive channels. However, principal issues about mechanosensitivity of aquaporins are not known. In this work, we characterized the mechanosensitive properties of the water channels BvTIP1;2 (TIP1) and BvPIP2;1 (PIP2) from red beet (Beta vulgaris). We simultaneously measured the mechanical behavior and the water transport rates during the osmotic response of emptied‐out oocytes expressing TIP1 or PIP2. Our results indicate that TIP1 is a mechanosensitive aquaporin, whereas PIP2 is not. We found that a single exponential function between the osmotic permeability coefficient and the volumetric elastic modulus governs the mechanosensitivity of TIP1. Finally, homology modeling analysis indicates that putative residues involved in mechanosensitivity show different quantity and distribution in TIP1 and PIP2.

The Water to Solute Permeability Ratio Governs the Osmotic Volume Dynamics in Beetroot Vacuoles

Plant cell vacuoles occupy up to 90% of the cell volume and, beyond their physiological function, are constantly subjected to water and solute exchange. The osmotic flow and vacuole volume dynamics relies on the vacuole membrane -the tonoplast- and its capacity to regulate its permeability to both water and solutes. The osmotic permeability coefficient (Pf) is the parameter that better characterizes the water transport when submitted to an osmotic gradient. Usually, Pf determinations are made in vitro from the initial rate of volume change, when a fast (almost instantaneous) osmolality change occurs. When aquaporins are present, it is accepted that initial volume changes are only due to water movements. However, in living cells osmotic changes are not necessarily abrupt but gradually imposed. Under these conditions, water flux might not be the only relevant driving force shaping the vacuole volume response. In this study, we quantitatively investigated volume dynamics of isolated Beta vulgaris root vacuoles under progressively applied osmotic gradients at different pH, a condition that modifies the tonoplast Pf. We followed the vacuole volume changes while simultaneously determining the external osmolality time-courses and analyzing these data with mathematical modeling. Our findings indicate that vacuole volume changes, under progressively applied osmotic gradients, would not depend on the membrane elastic properties, nor on the non-osmotic volume of the vacuole, but on water and solute fluxes across the tonoplast. We found that the volume of the vacuole at the steady state is determined by the ratio of water to solute permeabilites (Pf/Ps), which in turn is ruled by pH. The dependence of the permeability ratio on pH can be interpreted in terms of the degree of aquaporin inhibition and the consequently solute transport modulation. This is relevant in many plant organs such as root, leaves, cotyledons, or stems that perform extensive rhythmic growth movements, which very likely involve considerable cell volume changes within seconds to hours.