Gap junctions in the cardiovascular and immune systems

Gap junctions are clusters of intercellular channels directly connecting the cytoplasm of adjacent cells. These channels are formed by proteins named connexins and are present in all metazoan organisms where they serve diverse functions ranging from control of cell growth and differentiation to electric conduction in excitable tissues. In this overview we describe the presence of connexins in the cardiovascular and lympho-hematopoietic systems giving the reader a summary of the topics to be covered throughout this edition and a historical perspective of the discovery of gap junctions in the immune system.

Biophysical characteristics of gap junctions in vascular wall cells: implications for vascular biology and disease

The role gap junction channels play in the normal and abnormal functioning of the vascular wall is the subject of much research. The biophysical properties of gap junctions are an essential component in understanding how gap junctions function to allow coordinated relaxation and contraction of vascular smooth muscle. This study reviews the properties thus far elucidated and relates those properties to tissue function. We ask how biophysical and structural properties such as gating, permselectivity, subconductive states and channel type (heteromeric vs homotypic vs heterotypic) might affect vascular smooth muscle tone.

Gap junctions in isolated rat aorta: evidence for contractile responses that exhibit a differential dependence on intercellular communication

Connexin43 (Cx43) is a major gap junction protein present in the Fischer-344 rat aorta. Previous studies have identified conditions under which selective disruption of intercellular communication with heptanol caused a significant, readily reversible and time-dependent diminution in the magnitude of a1-adrenergic contractions in isolated rat aorta. These observations have indentified a significant role for gap junctions in modulating vascular smooth muscle tone. The goal of these steady-state studies was to utilize isolated rat aortic rings to further evaluate the contribution of intercellular junctions to contractions elicited by cellular activation in response to several other vascular spasmogens. The effects of heptanol were examined (0.2-2.0 mM) on equivalent submaximal (»75% of the phenylephrine maximum) aortic contractions elicited by 5-hydroxytryptamine (5-HT; 1-2 µM), prostaglandin F2a (PGF2a; 1 µM) and endothelin-1 (ET-1; 20 nM). Statistical analysis revealed that 200 µM and 500 µM heptanol diminished the maximal amplitude of the steady-state contractile responses for 5-HT from a control response of 75 ± 6% (N = 26 rings) to 57 ± 7% (N = 26 rings) and 34.9 ± 6% (N = 13 rings), respectively (P<0.05), and for PGF2a from a control response of 75 ± 10% (N = 16 rings) to 52 ± 8% (N = 19 rings) and 25.9 ± 6% (N = 18 rings), respectively (P<0.05). In contrast, 200 µM and 500 µM heptanol had no detectable effect on the magnitude of ET-1-induced contractile responses, which were 76 ± 5.0% for the control response (N = 38 rings), 59 ± 6.0% in the presence of 200 µM heptanol (N = 17 rings), and 70 ± 6.0% in the presence of 500 µM heptanol (N = 23 rings) (P<0.13). Increasing the heptanol concentration to 1 mM was associated with a significant decrease in the magnitude of the steady-state ET-1-induced contractile response to 32 ± 5% (21 rings; P<0.01); further increasing the heptanol concentration to 2 mM had no additional effect. In rat aorta then, junctional modulation of tissue contractility appears to be agonist-dependent.

Gap junctions in cells of the immune system: structure, regulation and possible functional roles

Gap junction channels are sites of cytoplasmic communication between contacting cells. In vertebrates, they consist of protein subunits denoted connexins (Cxs) which are encoded by a gene family. According to their Cx composition, gap junction channels show different gating and permeability properties that define which ions and small molecules permeate them. Differences in Cx primary sequences suggest that channels composed of different Cxs are regulated differentially by intracellular pathways under specific physiological conditions. Functional roles of gap junction channels could be defined by the relative importance of permeant substances, resulting in coordination of electrical and/or metabolic cellular responses. Cells of the native and specific immune systems establish transient homo- and heterocellular contacts at various steps of the immune response. Morphological and functional studies reported during the last three decades have revealed that many intercellular contacts between cells in the immune response present gap junctions or "gap junction-like" structures. Partial characterization of the molecular composition of some of these plasma membrane structures and regulatory mechanisms that control them have been published recently. Studies designed to elucidate their physiological roles suggest that they might permit coordination of cellular events which favor the effective and timely response of the immune system.

Gap junction modulation by extracellular signaling molecules: the thymus model

Gap junctions are intercellular channels which connect adjacent cells and allow direct exchange of molecules of low molecular weight between them. Such a communication has been described as fundamental in many systems due to its importance in coordination, proliferation and differentiation. Recently, it has been shown that gap junctional intercellular communication (GJIC) can be modulated by several extracellular soluble factors such as classical hormones, neurotransmitters, interleukins, growth factors and some paracrine substances. Herein, we discuss some aspects of the general modulation of GJIC by extracellular messenger molecules and more particularly the regulation of such communication in the thymus gland. Additionally, we discuss recent data concerning the study of different neuropeptides and hormones in the modulation of GJIC in thymic epithelial cells. We also suggest that the thymus may be viewed as a model to study the modulation of gap junction communication by different extracellular messengers involved in non-classical circuits, since this organ is under bidirectional neuroimmunoendocrine control.

Inhibition of return, gap effect and saccadic reaction time to a visual target

Simple manual reaction time (MRT) to a visual target (S2) is shortened when a non-informative cue (S1) is flashed at the S2 location shortly before the onset of S2 (early facilitation). Afterwards, MRT to S2 appearing at the S1 location is lengthened (inhibition of return - IOR). Similar results have been obtained for saccadic reaction time (SRT). Moreover, when there is a temporal gap between offset of the fixation point (FP) and onset of a target (gap paradigm), SRT is shorter than SRT in an overlap paradigm (FP remains on). In the present study, we determined SRT to S2 (10º) after presenting S1 at the same eccentricity (10º) or at a parafoveal position (2º) in the same or in the opposite hemifield. In addition, we employed both gap and overlap paradigms. Twelve subjects were asked not to respond to S1 (2º or 10º) to the right or to the left of FP, but to respond by making a saccadic movement in response to S2. We obtained the following results: 1) a 40-ms gap effect, 2) an interaction between gap effect and IOR, 3) a 39-ms delay (IOR) when S2 appeared at the cued (S1) position, and 4) a smaller (17 ms) but significant inhibition when S1 occurred at 2º in the ipsilateral hemifield. Thus, a parafoveal (2º) S1 elicits an inhibition of SRT towards ipsilateral peripheral targets. Since an inhibition of the ipsilateral hemifield by a 1º eccentric cue has been reported to occur when manual responses are employed, we suggest that the postulated functional link between covert and overt orienting of attention is also valid for parafoveal cues.

Looking for the GAP effect in manual responses and the role of contextual influences in reaction time experiments

When the offset of a visual stimulus (GAP condition) precedes the onset of a target, saccadic reaction times are reduced in relation to the condition with no offset (overlap condition) - the GAP effect. However, the existence of the GAP effect for manual responses is still controversial. In two experiments using both simple (Experiment 1, N = 18) and choice key-press procedures (Experiment 2, N = 12), we looked for the GAP effect in manual responses and investigated possible contextual influences on it. Participants were asked to respond to the imperative stimulus that would occur under different experimental contexts, created by varying the array of warning-stimulus intervals (0, 300 and 1000 ms) and conditions (GAP and overlap): i) intervals and conditions were randomized throughout the experiment; ii) conditions were run in different blocks and intervals were randomized; iii) intervals were run in different blocks and conditions were randomized. Our data showed that no GAP effect was obtained for any manipulation. The predictability of stimulus occurrence produced the strongest influence on response latencies. In Experiment 1, simple manual responses were shorter when the intervals were blocked (247 ms, P < 0.001) in relation to the other two contexts (274 and 279 ms). Despite the use of choice key-press procedures, Experiment 2 produced a similar pattern of results. A discussion addressing the critical conditions to obtain the GAP effect for distinct motor responses is presented. In short, our data stress the relevance of the temporal allocation of attention for behavioral performance.

Prolonged acceptance of skin grafts induced by B cells places regulatory T cells on the histopathology scene

The participation of regulatory T (Treg) cells in B cell-induced T cell tolerance has been claimed in different models. In skin grafts, naive B cells were shown to induce graft tolerance. However, neither the contribution of Treg cells to B cell-induced skin tolerance nor their contribution to the histopathological diagnosis of graft acceptance has been addressed. Here, using male C57BL/6 naive B cells to tolerize female animals, we show that skin graft tolerance is dependent on CD25+ Treg cell activity and independent of B cell-derived IL-10. In fact, B cells from IL-10-deficient mice were able to induce skin graft tolerance while Treg depletion of the host inhibited 100% graft survival. We questioned how Treg cell-mediated tolerance would impact on histopathology. B cell-tolerized skin grafts showed pathological scores as high as a rejected skin from naive, non-tolerized mice due to loss of skin appendages, reduced keratinization and mononuclear cell infiltrate. However, in tolerized mice, 40% of graft infiltrating CD4+ cells were FoxP3+ Treg cells with a high Treg:Teff (effector T cell) ratio (6:1) as compared to non-tolerized mice where Tregs comprise less than 8% of total infiltrating CD4 cells with a Treg:Teff ratio below 1:1. These results render Treg cells an obligatory target for histopathological studies on tissue rejection that may help to diagnose and predict the outcome of a transplanted organ.

Neuropeptide Y, substance P, and human bone morphogenetic protein 2 stimulate human osteoblast osteogenic activity by enhancing gap junction intercellular communication

Bone homeostasis seems to be controlled by delicate and subtle “cross talk” between the nervous system and “osteo-neuromediators” that control bone remodeling. The purpose of this study was to evaluate the effect of interactions between neuropeptides and human bone morphogenetic protein 2 (hBMP2) on human osteoblasts. We also investigated the effects of neuropeptides and hBMP2 on gap junction intercellular communication (GJIC). Osteoblasts were treated with neuropeptide Y (NPY), substance P (SP), or hBMP2 at three concentrations. At various intervals after treatment, cell viability was measured by the MTT assay. In addition, cellular alkaline phosphatase (ALP) activity and osteocalcin were determined by colorimetric assay and radioimmunoassay, respectively. The effects of NPY, SP and hBMP on GJIC were determined by laser scanning confocal microscopy. The viability of cells treated with neuropeptides and hBMP2 increased significantly in a time-dependent manner, but was inversely associated with the concentration of the treatments. ALP activity and osteocalcin were both reduced in osteoblasts exposed to the combination of neuropeptides and hBMP2. The GJIC of osteoblasts was significantly increased by the neuropeptides and hBMP2. These results suggest that osteoblast activity is increased by neuropeptides and hBMP2 through increased GJIC. Identification of the GJIC-mediated signal transduction capable of modulating the cellular activities of bone cells represents a novel approach to studying the biology of skeletal innervation.

Instrumental color and sensory acceptance of soy-based emulsions: a response surface approach

Response Surface Methodology (RSM) was applied to evaluate the chromatic features and sensory acceptance of emulsions that combine Soy Protein (SP) and red Guava Juice (GJ). The parameters analyzed were: instrumental color based on the coordinates a* (redness), b* (yellowness), L* (lightness), C* (chromaticity), h* (hue angle), visual color, acceptance, and appearance. The analyses of the results showed that GJ was responsible for the high measured values of red color, hue angle, chromaticity, acceptance, and visual color, whereas SP was the variable that increased the yellowness intensity of the assays. The redness (R²adj = 74.86%, p < 0.01) and hue angle (R²adj = 80.96%, p < 0.01) were related to the independent variables by linear models, while the sensory data (color and acceptance) could not be modeled due to a high variability. The models of yellowness, lightness, and chromaticity did not present lack of fit but presented adjusted determination coefficients bellow 70%. Notwithstanding, the linear correlations between sensory and instrumental data were not significant (p > 0.05) and low Pearson coefficients were obtained. The results showed that RSM is a useful tool to develop soy-based emulsions and model some chromatic features of guava-based emulsions through RSM.

Concentration of pineapple juice by reverse osmosis: physicochemical characteristics and consumer acceptance

Reverse osmosis has been used for the concentration of fruit juices with promising considering the quality of the obtained products. The objective of this study was to concentrate single strength pineapple juice by reverse osmosis. The concentration was carried out with polyamide composite membranes in a 0.65 m² plate and frame module at 60 bar transmembrane pressure at 20 °C. The permeate flux was 17 L.hm-2. The total soluble solid content of the juice increased from 11 to 31 °Brix corresponding to a Volumetric Concentration Factor (VCF) of 2.9. The concentration of soluble solids, total solids, and total acidity increased proportionally to FCV. The concentrated juice and three commercial concentrated pineapple juices were evaluated regarding preference and purchase intention by 79 pineapple juice consumers. The concentrated juice by reverse osmosis was the preferred among consumers. It can be concluded that this process may be considered an alternative to the pre-concentration of fruit juices.

Influence of color on acceptance and identification of flavor of foods by adults

The sensory characteristics color and flavor of food play an important role not only in the selection, but also in the determination of consumption, satiation, and ingestion. With the objective to determine and evaluate the influence of color on the acceptance and identification of flavor of foods for adults, sensory analysis was performed on jellies by non-trained tasters of both sexes aged between 18 and 60 years (1750 tests). A hedonic scale and combinations of five colors (red, yellow, green, blue and purple) and three flavors (strawberry, pineapple, and limes) were used in the acceptance test totaling 15 samples. In the duo-trio discrimination test, together with the reference sample (R), one sample identical to the reference and another of identical color and different flavor were offered, and the judges were requested to identify the sample that was different from the reference sample. The colors used did not influence the acceptance of the samples (P > 0.05), and as there was not significant interaction between color and flavor. However, the limes flavor negatively influenced acceptance when compared to the other flavors. With regard to flavor differentiation, the colors used did not influence flavor identification (P > 0.05); However, differentiated behavior was identified between females and males, and the latter were more error-prone. Therefore, under the experimental conditions tested, color did not influence the acceptance and identification of the flavor of the samples by adults.

Ordered probit regression analysis of the effect of brand name on beer acceptance by consumers

Ordered probit regression was used to analyze data of sensory acceptance tests designed to study the effect of brand name on the acceptability of beer samples. Eight different brands of Pilsen beer were evaluated by 101 consumers in two sessions of acceptance tests: blind evaluation and brand information test. Ordered probit regression, although a relatively sophisticated technique compared to others used to analyze sensory data, was chosen to enable the observation of consumers' behavior using graphical interpretations of estimated probabilities plotted against hedonic scales. It can be concluded that brands B, C, and D had a positive effect on the sensory acceptance of the product, whereas brands A, F, G, and H had a negative influence on consumers' evaluation of the samples. On the other hand, brand E had little influence on consumers' assessment.

Transference of lutein during cheese making, color stability, and sensory acceptance of prato cheese

The consumption of lutein is associated with the prevention and reduction of age-related macular degeneration. Its incorporation into Prato cheese as a yellowish food coloring is a valid alternative to increase the daily intake of this compound. However, part of the lutein added may be lost in the whey during the cheese making, or it can be degraded by light during storage, resulting in color changes reducing the sensory acceptance of the cheese. The objectives of this study were to determine the transference of the lutein (dye), added to the milk, in the whey, and cheese, to evaluate the effect of the lutein addition, light exposure, and storage time on the cheese color, and to verify the sensory acceptance of Prato cheese with addition of lutein. The lutein recovery of cheese was 95.25%. Color saturation (chrome) increased during storage time resulting in a cheese with more intense color, but there were no changes in the hue of the cheese. Adjusting the amount of lutein added to Prato cheese may lead to greater acceptance. The high recovery of lutein in the cheese and the fact that the hue remained unchanged during storage under light showed that the incorporation of lutein into Prato cheese is feasible from a technical point of view.

Acceptance of nanotechnology applications and satisfaction with food-related life in southern Chile

Given the increasing use of nanotechnology in food production and packaging, its acceptance was evaluated in Temuco, Chile, and different consumer segments were identified. Different brands of sunflower oil were used at different prices as a case study. A structured questionnaire was applied to 400 supermarket shoppers. It was determined that brand was more important than nanotechnology application in packaging and food, and more important than price. The consumers preferred an average priced oil with a manufacturer's brand with nanoparticles to reduce cholesterol, and packaging with nanoparticles to increase the shelf life of the product and to prevent the growth of microorganisms. Three consumer segments were distinguished by the cluster analysis. The largest segment (44%) preferred the oil without nanotechnology. The second (35.2%) preferred the oil with nanotechnology in the food and the packaging, and the greatest preference was for packaging with nanoparticles extension in the shelf life of the product. The third segment (20.8%) had similar behavior, but it showed greater preference for the oil with nanoparticles that reduced cholesterol and for the packaging that prevented the growth of bacteria and viruses. The segments differed in terms of their satisfaction with food-related life and lifestyle. It was found that over 50% of the participants preferred oil with nanotechnology applications.