Biomedical subjects

P R Brink

Publications and source records attributed to P R Brink.

At least 19 recordsLinked to original sources

PubMed2005

High-resolution imaging characterization of bladder dynamic morphophysiology by time-lapse optical coherence tomography.

We report an experimental study of the possibility of high-speed optical coherence tomography (OCT) for high-resolution imaging characterization of detrusor dynamic morphophysiology and analysis of the mechanisms that lead to geriatric incontinence (GI). The spontaneous contractility of intact fresh rabbit bladders was imaged with two-dimensional (2D) OCT ex vivo at up to 8 frames/s. The time-lapse 2D OCT images were postprocessed by image segmentation and fast-Fourier-transform analysis to characterize the dynamic morphological changes of the bladder contractility. In addition, we studied young and aging rat bladders to analyze the differences in dynamics. Preliminary results of our ex vivo study reveal that time-lapse OCT can track the contractile waves of bladders at high spatial resolution and characterize their dynamic morphophysiology in terms of amplitude, phase, and frequency. The results suggest that time-lapse OCT has the potential to act as a detrusor optical biopsy to enhance the diagnosis of detrusor dysfunction and thus of the mechanisms that lead to GI.

Algorithms↗

PubMed2005

Connexin-specific cell-to-cell transfer of short interfering RNA by gap junctions.

The purpose of this study was to determine whether oligonucleotides the size of siRNA are permeable to gap junctions and whether a specific siRNA for DNA polymerase beta (pol beta) can move from one cell to another via gap junctions, thus allowing one cell to inhibit gene expression in another cell directly. To test this hypothesis, fluorescently labelled oligonucleotides (morpholinos) 12, 16 and 24 nucleotides in length were synthesized and introduced into one cell of a pair using a patch pipette. These probes moved from cell to cell through gap junctions composed of connexin 43 (Cx43). Moreover, the rate of transfer declined with increasing length of the oligonucleotide. To test whether siRNA for pol beta was permeable to gap junctions we used three cell lines: (1) NRK cells that endogenously express Cx43; (2) Mbeta16tsA cells, which express Cx32 and Cx26 but not Cx43; and (3) connexin-deficient N2A cells. NRK and Mbeta16tsA cells were each divided into two groups, one of which was stably transfected to express a small hairpin RNA (shRNA), which gives rise to siRNA that targets pol beta. These two pol beta knockdown cell lines (NRK-kcdc and Mbeta16tsA-kcdc) were co-cultured with labelled wild type, NRK-wt or Mbeta16tsA-wt cells or N2A cells. The levels of pol beta mRNA and protein were determined by semiquantitative RT-PCR and immunoblotting. Co-culture of Mbeta16tsA-kcdc cells with Mbeta16tsA-wt, N2A or NRK-wt cells had no effect on pol beta levels in these cells. Similarly, co-culture of NRK-kcdc with N2A cells had no effect on pol beta levels in the N2A cells. In contrast, co-culture of NRK-kcdc with NRK-wt cells resulted in a significant reduction in pol beta in the wt cells. The inability of Mbeta16tsA-kcdc cells to transfer siRNA is consistent with the fact that oligonucleotides of the 12 nucleotide length were not permeable to Cx32/Cx26 channels. This suggested that Cx43 but not Cx32/Cx26 channels allowed the cell-to-cell movement of the siRNA. These results support the novel hypothesis that non-hybridized and possible hybridized forms of siRNA can move between mammalian cells through connexin-specific gap junctions.

Animals↗

PubMed2005

Non-stationary fluctuation analysis of macroscopic gap junction channel records.

Non-stationary fluctuation analysis was applied to macroscopic records of junctional currents arising from homotypic Cx37 and Cx43 gap junction channels expressed in RIN cells. The data were analyzed by a modification of existing analytical methods that takes endemic uncoupling into account. The results are consistent with both channels having open probabilities ranging from 0.7 to near unity for low transjunctional voltages. The analysis also yielded estimates of single-channel conductances for the two channel types similar to those seen in single-channel recordings. The results presented here show that fluctuation analysis can be used to extract single-channel gap junctional conductances from macroscopic double whole-cell recordings. These results also constitute empirically determined estimates of the open probability that are not model-dependent.

Animals↗

PubMed2001

Site-directed mutations in the transmembrane domain M3 of human connexin37 alter channel conductance and gating.

Connexin37 (Cx37) is expressed principally in endothelial cells. We have introduced individual point mutations (Cx37-V156D or Cx37-K162E) in the putative pore lining segment M3 of a polymorphic human Cx37 (Cx37-S319) and expressed them in N2A and RIN cells. RT-PCR and immunofluorescence microscopy were used to confirm the expression of the proteins. Stably transfected cells were subjected to electrophysiological studies. Experiments were performed on cell pairs using the dual whole cell patch-clamp method. Single channel records showed that both mutants display a variety of conductive states (Cx37-V156D, 47-250 pS; Cx37-K162E, 58-342 pS) in contrast to the typical high conductance of 340-375 pS and subconductive state of 60-80 pS reported for Cx37-S319. Analysis of the macroscopic data for Cx37-K162E revealed a broadened Vo indicating the influence of the mutation on voltage gating. Our data indicate that substitution of a conserved residue with a charged residue could cause changes in the main state and/or in the size of the pore. It is possible that these particular residues in the M3 domain interact electrostatistically with several of the other domains in the Cx37 protein.

Animals↗

PubMed2001

Comparative clinical trial of progressive dilatational and forceps dilatational tracheostomy.

OBJECTIVE: To compare the safety and early complications of progressive dilatational tracheostomy (PDT) and forceps dilatational tracheostomy (FDT). DESIGN: Prospective randomized trial. SETTING: Three-centre university and public teaching hospitals. PATIENTS: One hundred and twenty-seven consecutive patients were randomized to undergo PDT or FDT. RESULTS: The duration of the procedure was 7.0 +/- 3.5 min in the PDT group and 6.4 +/- 4.9 min in the FDT group, which was not significantly different. Intraprocedural complications occurred in 14 % with PDT and 31% with FDT (p = 0.03), and were usually minor. Difficult or false insertion of the cannula in eight patients after FDTwas the most common complication. CONCLUSIONS: Both percutaneous tracheostomy techniques are safe. The early complication rate of PDT appeared to be lower than FDT, but the early complication rate of FDT may be decreased significantly with small modifications to the set.

Aged↗

PubMed2001

Lacrimal gland fluid secretion and lymphocytic infiltration in the NZB/W mouse model of Sjögren's syndrome.

PURPOSE: The fluid secretory impairment of lacrimal and salivary glands in Sjögren's syndrome (SS) is thought to be related to the extent of lymphocytic infiltration (LI) and subsequent loss of glandular tissue. In this study, we examine the correlation between the extent of tear flow reduction and the extent of LI of lacrimal glands in the NZB/W mouse, a model of SS. METHODS: We stimulated tear production by topical application of carbachol onto the gland while fluid was collected from the lacrimal duct. The lacrimal glands were removed after fluid collection for histology. RESULTS: Fluid secretion in response to carbachol was less in the majority of young NZB/W females compared to C57 control animals and none of the glands showed LI. Fluid secretion was also impaired in the majority of old NZB/W females, and the extent of LI was highly variable. Some of the old SW females also showed blunted fluid secretory responses and some degree of focal LI. Young SW females showed no LI and most animals exhibited normal flow responses. Analysis of paired flow and LI measurements showed no correlation between LI and flow impairment in any of the groups or in the pooled data. Carbachol-stimulated protein secretion from lacrimal gland slices in vitro were similar in young and old SW and NZB/W mice. CONCLUSIONS: These results suggest that LI alone is not sufficient to explain the secretory dysfunction in the NZB/W mouse model of Sjögren's syndrome.

Administration, Topical↗

PubMed2001

Heteromeric connexons formed by the lens connexins, connexin43 and connexin56.

In the eye lens, three connexins have been detected in epithelial cells and bow region/differentiating fiber cells, suggesting the possible formation of heteromeric gap junction channels. To study possible interactions between Cx56 and Cx43, we stably transfected a normal rat kidney cell line (NRK) that expresses Cx43 with Cx56 (NRK-Cx56). Similar to the lens, several bands of Cx56 corresponding to phosphorylated forms were detected by immunoblotting in NRK-Cx56 cells. Immunofluorescence studies showed co-localization of Cx56 with Cx43 in the perinuclear region and at appositional membranes. Connexin hexamers in NRK-Cx56 cells contained both Cx43 and Cx56 as demonstrated by sedimentation through sucrose gradients. Immunoprecipitation of Cx56 from sucrose gradient fractions resulted in co-precipitation of Cx43 from NRK-Cx56 cells suggesting the presence of relatively stable interactions between the two connexins. Double whole-cell patch-clamp experiments showed that the voltage-dependence of Gmin in NRK-Cx56 cells differed from that in NRK cells. Moreover, stable interactions between Cx43 and Cx56 were also demonstrated in the embryonic chicken lens by co-precipitation of Cx43 in Cx56 immunoprecipitates. These data suggest that Cx43 and Cx56 form heteromeric connexons in NRK-Cx56 cells as well as in the lens in vivo leading to differences in channel properties which might contribute to the variations in gap junctional intercellular communication observed in different regions of the lens.

Animals↗

PubMed2001

Intercellular communication in cultured human vascular smooth muscle cells.

Intercellular communication through gap junction channels plays a fundamental role in regulating vascular myocyte tone. We investigated gap junction channel expression and activity in myocytes from the physiologically distinct vasculature of the human internal mammary artery (IMA, conduit vessel) and saphenous vein (SV, capacitance vessel). Northern and Western blots documented the presence of connexin43 (Cx43) in frozen tissues and cultured cells from both vessels. Northern blots also confirmed the presence of Cx40 mRNA in cultured IMA and SV myocytes. Dual whole cell patch-clamp experiments revealed that macroscopic junctional conductance was voltage dependent and characteristic of that observed for Cx43. In the majority of records, in both vessels, single-channel activity was dominated by a main-state conductance of 120 pS, with subconducting events comprising less than 10% of the amplitude histograms. However, some records showed "atypical" unitary events that had a conductance similar to Cx40 (approximately 140-160 pS), but gating behavior like that of Cx43. As such, it is conceivable that the presence and coexpression of Cx40 and Cx43 in IMA and SV myocytes may result in heteromeric channel formation. Nonetheless, in terms of gating, Cx43-like behavior clearly dominates.

Aged↗

PubMed2001

Gap junction channels formed by coexpressed connexin40 and connexin43.

Many cardiovascular cells coexpress multiple connexins (Cx), leading to the potential formation of mixed (heteromeric) gap junction hemichannels whose biophysical properties may differ from homomeric channels containing only one connexin type. We examined the potential interaction of connexin Cx43 and Cx40 in HeLa cells sequentially stably transfected with these two connexins. Immunoblots verified the production of comparable amounts of both connexins, cross-linking showed that both connexins formed oligomers, and immunofluorescence showed extensive colocalization. Moreover, Cx40 copurified with (His)(6)-tagged Cx43 by affinity chromatography of detergent-solubilized connexons, demonstrating the presence of both connexins in some hemichannels. The dual whole cell patch-clamp method was used to compare the gating properties of gap junctions in HeLa Cx43/Cx40 cells with homotypic (Cx40-Cx40 and Cx43-Cx43) and heterotypic (Cx40-Cx43) gap junctions. Many of the observed single channel conductances resembled those of homotypic or heterotypic channels. The steady-state junctional conductance (g(j,ss)) in coexpressing cell pairs showed a reduced sensitivity to the voltage between cells (V(j)) compared with homotypic gap junctions and/or an asymmetrical V(j) dependence reminiscent of heterotypic gap junctions. These gating properties could be fit using a combination of homotypic and heterotypic channel properties. Thus, whereas our biochemical evidence suggests that Cx40 and Cx43 form heteromeric connexons, we conclude that they are functionally insignificant with regard to voltage-dependent gating.

Animals↗

PubMed2001

Heteromeric mixing of connexins: compatibility of partners and functional consequences.

Cx43 is widely expressed in many different cell types, and many of these cells also express other connexins. If these connexins are capable of mixing, the functional properties of channels containing heteromeric connexons may substantially influence intercellular communication between such cells. We used biochemical strategies (sedimentation through sucrose gradients, co-immunoprecipitation, or co-purification by Ni-NTA chromatography) to examine heteromeric mixing of Cx43 with other connexins (including Cx26, Cx37, Cx40, Cx45, and Cx56) in transfected cells. These analyses showed that all of the tested connexins except Cx26 formed heteromeric connexons with Cx43. We used the double whole-cell patch-camp technique to analyze the electrophysiological properties of gap junction channels in pairs of co-expressing cells. Cx37 and Cx45 made a large variety of functional heteromeric combinations with Cx43 based on detection of many different single channel conductances. Most of the channel event sizes observed in cells co-expressing Cx40 and Cx43 were similar to those of homomeric Cx43 or Cx40 hemichannels in homo- or hetero-typic configurations. Our data suggest several different possible consequences of connexin co-expression: (1) some combinations of connexins may form heteromeric connexons with novel proeprties; (2) some connexins may form heteromeric channels that do not have unique properties, and (3) some connexins may be incompatible for heteromeric mixing.

Animals↗

PubMed2000

Functional expression and biophysical properties of polymorphic variants of the human gap junction protein connexin37.

Connexin37 (Cx37) forms gap junction channels between endothelial cells, and two polymorphic Cx37 variants (Cx37-S319 and Cx37-P319) have been identified with a possible link to atherosclerosis. We studied the gap junction channel properties of these hCx37 polymorphs by expression in stably transfected communication-deficient cells (N2A and RIN). We also expressed a third, truncated variant (Cx37-fs254Delta293) and Cx37 constructs containing epitope tags added to their amino or carboxyl termini. All Cx37 constructs were produced by the transfected cells as demonstrated by RT-PCR and immunoblotting and trafficked to appositional surfaces between cells as demonstrated by immunofluorescence microscopy. Dual whole cell patch-clamping studies demonstrated that Cx37-P319, Cx37-S319, and Cx37-fs254Delta293 had large unitary conductances ( approximately 300 pS). However, addition of an amino terminal T7 tag (T7-Cx37-fs254Delta293) produced a single channel conductance of 120-145 pS with a 24-30 pS residual state. Moreover, the kinetics of the voltage-dependent decline in junctional current for T7-Cx37-fs254Delta293 were significantly slower than for the wild type, implying a destabilization of the transition state. These data suggest that the amino terminus of Cx37 plays a significant role in gating as well as conductance. The carboxyl terminal tail has lesser influence on unitary conductance and inactivation kinetics.

Amino Acid Sequence↗

PubMed2000

Formation of heterotypic gap junction channels by connexins 40 and 43.

Gap junctions formed between transfected cells expressing connexin (Cx) 40 and Cx43 (Cx43-RIN, Cx40-HeLa, and Cx43-HeLa) revealed a relationship, g(j)=f(V(j)), at steady state, that is typified by a nonsymmetrical behavior similar to that previously reported for other heterotypic channels (gap junction conductance [g(j)]; transjunctional voltage [V(j)]). The unitary conductance of the channels was sensitive to the polarity of V(j). A main state conductance of 61 pS was found when the Cx43 cell was stepped positively or the Cx40 cell negatively (V(j)=70 mV); the reverse polarities yielded a conductance of 100 pS. These heterotypic channels were permeable to carboxyfluorescein. In addition, two other heterotypic forms are illustrated to demonstrate that endogenous Cx45 expression cannot explain the results. The demonstration of heterotypic Cx40-Cx43 channels may have implications for the propagation of the electrical impulse in heart. For example, they may contribute to the slowing of the impulse propagation through the junctions between Purkinje fibers and ventricular muscle.

Animals↗

PubMed2000

The influence of surface charges on the conductance of the human connexin37 gap junction channel.

The single-channel conductance of the hCx37 homotypic gap junction channel does not saturate with transjunctional voltages up to +/-75 mV, nor does it depend linearly on the intracellular electrolyte concentration. The average maximum unitary conductances measured in KCl were 175 pS (30 mM), 236 pS (55 mM), 343 pS (110 mM), and 588 pS (270 mM) in the presence of 0.1 mM MgCl(2). The unexpectedly high unitary conductance at low salt concentrations can be explained by fixed charge groups within or near the channel orifice. Fixed cytoplasmic surface charges (3.4 e) positioned adjacent (15 A) to the channel pore adequately model the data (surface charge density of 0.24 e/(nm)(2)). In other experiments, high Mg(2+) reduced the unitary conductance of hCx37 homotypic gap junction channels more than predicted by screening alone, consistent with specific effects of Mg(2+) on the channel.

Connexins↗

PubMed2000

Modulation of intercellular communication by differential regulation and heteromeric mixing of co-expressed connexins.

Intercellular communication may be regulated by the differential expression of subunit gap junction proteins (connexins) which form channels with differing gating and permeability properties. Endothelial cells express three different connexins (connexin37, connexin40, and connexin43) in vivo. To study the differential regulation of expression and synthesis of connexin37 and connexin43, we used cultured bovine aortic endothelial cells which contain these two connexins in vitro. RNA blots demonstrated discordant expression of these two connexins during growth to confluency. RNA blots and immunoblots showed that levels of these connexins were modulated by treatment of cultures with transforming growth factor-ss1. To examine the potential ability of these connexins to form heteromeric channels (containing different connexins within the same hemi-channel), we stably transfected connexin43-containing normal rat kidney (NRK) cells with connexin37 or connexin40. In the transfected cells, both connexin proteins were abundantly produced and localized in identical distributions as detected by immunofluorescence. Double whole-cell patch-clamp studies showed that co-expressing cells exhibited unitary channel conductances and gating characteristics that could not be explained by hemi-channels formed of either connexin alone. These observations suggest that these connexins can readily mix with connexin43 to form heteromeric channels and that the intercellular communication between cells is determined not only by the properties of individual connexins, but also by the interactions of those connexins to form heteromeric channels with novel properties. Furthermore, modulation of levels of the co-expressed connexins during cell proliferation or by cytokines may alter the relative abundance of different heteromeric combinations.

Animals↗

PubMed2000

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.

Biophysical Phenomena↗

PubMed2000

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 alpha1-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 ( approximately 75% of the phenylephrine maximum) aortic contractions elicited by 5-hydroxytryptamine (5-HT; 1-2 microM), prostaglandin F2alpha (PGF2alpha; 1 microM) and endothelin-1 (ET-1; 20 nM). Statistical analysis revealed that 200 microM and 500 microM 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 PGF2alpha 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 microM and 500 microM 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 microM heptanol (N = 17 rings), and 70 +/- 6.0% in the presence of 500 microM 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.

Animals↗

PubMed1999

[Penetrating heart injuries in two butchers].

Two butchers, men aged 25 and 48 years, sustained a perforating heart injury when they made a vertical cut. They were rushed to hospital. The first patient was haemodynamically stable on arrival. The second had hypotension and cardiac ultrasonography revealed increased fluid in the pericardium; he was subjected to pericardiocentesis. The heart wounds in both patients were sutured via a left-sided anterolateral thoracotomy; the younger had a wound in the left ventricle, the older both a dorsal and a ventral transmural perforation of the right ventricle. Both patients could be discharged after a little over a week in good condition. Employers in abbattoirs should provide protective clothing fastening high enough to prevent heart wounds.

Abattoirs↗

PubMed1999

Analysis of the presence and physiological relevance of subconducting states of Connexin43-derived gap junction channels in cultured human corporal vascular smooth muscle cells.

Subconductance states are a commonly observed feature of gap junction channels. Their overt frequency and consistent appearance in both single and multichannel records have led to speculation that they might be of physiological importance in terms of altering the rate of small solute transfer from cell to cell. Among the connexin gene family, connexin43 (Cx43) is the most ubiquitous connexin that has been shown to generate subconductive states. Therefore, it was the explicit aim of this investigation to more fully evaluate the potential contribution of human Cx43-derived subconducting states to intercellular communication in cultured human corporal vascular smooth muscle cells. To determine the weight of subconductive states in our records, we analyzed amplitude histograms of multichannel and single-channel data during the application of transjunctional voltages larger than expected for physiological conditions but still smaller than transjunctional voltages known to induce lower conductive states (Vo>Vj). The data clearly indicated that the subconducting states occupy only a small fraction of the total channel open time. This was reflected by the fact that the average open probability for the subconductive state(s) determined from the 9 records analyzed was approximately 2%. Closer inspection of the data revealed that the frequency of subconductive states was actually higher than the frequency of the main state conductance. In summary, recording conditions sufficient for evaluation of the intrinsic gating characteristics of human Cx43-derived gap junction channels have been used. Under these conditions, our data clearly indicate that despite their greater frequency, the duration of subconductance events is so short relative to the main state duration as to render them physiologically insignificant.

Cells, Cultured↗