Kyung Hwan Seul

Evidence for heteromeric gap junction channels formed from rat connexin43 and human connexin37

Homomeric gap junction channels are composed solely of one connexin type, whereas heterotypic forms contain two homomeric hemichannels but the six identical connexins of each are different from each other. A heteromeric gap junction channel is one that contains different connexins within either or both hemichannels. The existence of heteromeric forms has been suggested, and many cell types are known to coexpress connexins. To determine if coexpressed connexins would form heteromers, we cotransfected rat connexin43 (rCx43) and human connexin37 (hCx37) into a cell line normally devoid of any connexin expression and used dual whole cell patch clamp to compare the observed gap junction channel activity with that seen in cells transfected only with rCx43 or hCx37. We also cocultured cells transfected with hCx37 or rCx43, in which one population was tagged with a fluorescent marker to monitor heterotypic channel activity. The cotransfected cells possessed channel types unlike the homotypic forms of rCx43 or hCx37 or the heterotypic forms. In addition, the noninstantaneous transjunctional conductance-transjunctional voltage ( G j/ V j) relationship for cotransfected cell pairs showed a large range of variability that was unlike that of the homotypic or heterotypic form. The heterotypic cell pairs displayed asymmetric voltage dependence. The results from the heteromeric cell pairs are inconsistent with summed behavior of two independent homotypic populations or mixed populations of homotypic and heterotypic channels types. The G j/ V jdata imply that the connexin-to-connexin interactions are significantly altered in cotransfected cell pairs relative to the homotypic and heterotypic forms. Heteromeric channels are a population of channels whose characteristics could well impact differently from their homotypic counterparts with regard to multicellular coordinated responses.Homomeric gap junction channels are composed solely of one connexin type, whereas heterotypic forms contain two homomeric hemichannels but the six identical connexins of each are different from each other. A heteromeric gap junction channel is one that contains different connexins within either or both hemichannels. The existence of heteromeric forms has been suggested, and many cell types are known to coexpress connexins. To determine if coexpressed connexins would form heteromers, we cotransfected rat connexin43 (rCx43) and human connexin37 (hCx37) into a cell line normally devoid of any connexin expression and used dual whole cell patch clamp to compare the observed gap junction channel activity with that seen in cells transfected only with rCx43 or hCx37. We also cocultured cells transfected with hCx37 or rCx43, in which one population was tagged with a fluorescent marker to monitor heterotypic channel activity. The cotransfected cells possessed channel types unlike the homotypic forms of rCx43 or hCx37 or the heterotypic forms. In addition, the noninstantaneous transjunctional conductance-transjunctional voltage (Gj/Vj) relationship for cotransfected cell pairs showed a large range of variability that was unlike that of the homotypic or heterotypic form. The heterotypic cell pairs displayed asymmetric voltage dependence. The results from the heteromeric cell pairs are inconsistent with summed behavior of two independent homotypic populations or mixed populations of homotypic and heterotypic channels types. The Gj/Vj data imply that the connexin-to-connexin interactions are significantly altered in cotransfected cell pairs relative to the homotypic and heterotypic forms. Heteromeric channels are a population of channels whose characteristics could well impact differently from their homotypic counterparts with regard to multicellular coordinated responses.

Extracellular fluid translocation in perfused rabbit atria: implication in control of atrial natriuretic peptide secretion.

1. Transmural transport of 22Na+, 51Cr‐EDTA, [3H]inulin and [14C]Dextran (57 kDa) was measured in perfused rabbit atria. The radiolabelled extracellular space (ECS) markers and [14C]Dextran were introduced into the pericardial space or atrial lumen. Atrial volume changes were induced by steps up and down in atrial pressure. 2. Basal rates of transmural transport of radiolabelled ECS markers across the atrial wall were relatively stable up to 70 min. Atrial stretch and release resulted in a rapid but transient, and reversible increase in the ECS fluid (ECF) translocation. The increased translocation of the ECF into the atrial lumen occurred within 15 s of the reduction of atrial distension and returned to the baseline level within 60 s. 3. Transmural transport of [3H]inulin across the atrial wall was bidirectional. 4. The clearance of radiolabelled ECS markers was molecular‐size dependent. The transmural clearance of [3H]inulin was dependent on the distension‐reduction volume changes induced by atrial stretch and release. Little transport of [14C]Dextran across the atrial wall was observed. 5. The ECF translocation across the atrial wall was not influenced by changes in external Ca2+ but was suppressed by low temperature. 6. Dynamic changes in the ECS of the atrium were observed in response to atrial distension and reduction. The ECS of the atrium increased on distension and decreased on reduction of atrial distension. 7. Reduction in atrial distension resulted in an increase in the secretion of immunoreactive atrial natriuretic peptide (ANP) which coincided with an increase in the translocation of the ECF. The secretion of immunoreactive ANP was a function of the translocation of the ECF. 8. It is suggested that atrial stretch and release may play a role in driving fluid flow within the interstitium and fluid translocation out of the interstitium. This fluid movement presumably leads to convective transport of released ANP into the atrial lumen.

Presence of immunoreactive atrial natriuretic peptide in follicular fluid, ovary and ovarian perfusates

Immunoreactive atrial natriuretic peptide (ir-ANP) was measured in the follicular fluid of pig ovarian follicle, and rabbit ovarian homogenates and perfusates using a specific radioimmunoassay (RIA). Serial dilution curves made with the extracts of follicular fluid, ovarian homogenates and perfusates using SepPak C18 cartridges were parallel with the RIA standard curve. On gel filtration chromatography and reverse phase HPLC, all extracted materials showed high and low molecular weight forms of ir-ANP. The amount of ir-ANP in rabbit ovary was 40.70 +/- 0.39 pg/mg and that in follicular fluid of pig ovarian follicle was 18.88 +/- 2.49 pg/ml.

Mechanical Control of Extracellular Space in Rabbit Atria: An Intimate Modulator of the Translocation of Extracellular Fluid and Released Atrial Natriuretic Peptide

We have previously shown that extracellular fluid (ECF) is translocated by atrial contraction. Following on from this finding we have proposed a two‐step sequential mechanism for the regulation of stretch‐activated secretion of atrial natriuretic peptide (ANP): myocytic release of ANP into the surrounding paracellular space followed by the translocation of ECF with the released ANP into the bloodstream. This latter step is induced by atrial contraction, and is therefore controlled by atrial workload. However, the mechanism that regulates the changes in translocation of the ECF has not been defined. To define the relationship between the atrial workload, ECF translocation, size of the extracellular space (ECS) and ANP secretion, experiments have been performed in isolated perfused beating rabbit atria. Atrial workload, transendocardial translocation of the ECF and the secretion of ANP were quantified. Changes in the size of the atrium and the ECS were determined by a newly developed methodology in the beating atria. Atrial workload determined the translocation of the ECF and released ANP with waning of the translocation at higher myocardial workloads. Atrial workload inversely determined the size of the atrium and the ECS. The latter directly determined the translocation of the ECF in terms of atrial workload. From these data we suggest that the size of the ECS is an intimate modulator of the translocation of the ECF and released ANP, and that the phenomenon of waning of the transendocardial translocation that appeared at higher atrial workloads is closely related to the shrinkage of the ECS.

Plasma Concentration of Atrial Natriuretic Peptide in Different Phases of Korean Hemorrhagic Fever

Korean hemorrhagic fever (KHF) is an epidemic viral disease characterized by high fever, hemorrhagic tendency and renal failure, and by hemorrhages of right atrium and renal medulla as well as necrosis of anterior hypophysis. Plasma immunoreactive atrial natriuretic peptide (irANP) levels of 15 patients in the oliguric phase was 94.8 +/- 8.4 pg/ml (mean +/- SEM), 80% higher than of the normal control group (53.0 +/- 4.7 pg/ml; n = 28). In the diuretic phase it declined to 63.7 +/- 5.3 pg/ml (n = 26). Plasma renin activity (PRA) in the oliguric phase was 19.0 +/- 1.3 ng AI/ml/h, and in the diuretic phase 5.3 +/- 0.9 ng AI/ml/h, significantly higher than the control value (2.5 +/- 0.1 ng AI/ml/h). Elevations of irANP and PRA were not correlated in each group. Also systemic blood pressure as well as heart beats were significantly increased in the oliguric phase. These findings suggest that the increased irANP may have resulted from increased circulatory volume and that the ANP secretory process may not be affected by the disease.

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.

Reduction volume dependence of immunoreactive atrial natriuretic peptide secretion in isolated perfused rabbit atria

A new technique to permit gradual changes in atrial distension has been developed in an isolated perfused rabbit atrium preparation. Graded volume reduction in the atrium was induced by changing the elevation of the outflow catheter tip. Pressure reduction from 6 cm H2O atrial distension resulted in a decrease in atrial distension volume. Atrial distension by 6 cmH2O did not change the release of immunoreactive atrial natriuretic peptide (irANP). The graded reduction in atrial distension from 0.11 +/- 0.03 (1.5 cm H2O) to 1.36 +/- 0.19 microliters/mg wet weight (6.0 cm H2O) resulted in 1.7 (6.76 +/- 2.05 versus 3.83 +/- 1.18 pg/mg per min, n = 9, P less than 0.025) to 40.1-fold (77.66 +/- 17.82 versus 3.0 +/- 1.14 pg/mg per min, n = 11, P less than 0.025) increases in irANP release. IrANP release in response to the reduction of atrial distension was volume dependent. The relation of percentage increase in irANP release with the percentage reduction of atrial distension was exponential. The data suggest that the atrial muscle shortening, but not stretch per se, may be a potent direct stimulus for the regulation of irANP secretion.

SEQUENTIAL MECHANISM OF ATRIAL-NATRIURETIC-PEPTIDE SECRETION IN ISOLATED PERFUSED RABBIT ATRIA

It is well known that the secretion of atrial natriuretic peptide (ANP) is dependent on the atrial stretch. It has been claimed in this laboratory that the secretion of ANP occurs with a reduction in atrial distension. It was shown in the present experiment that the secretion of immunoreactive (ir) ANP occurs coincidently with a translocation of extracellular space marker (3-H)-inulin in the isolated perfused rabbit atria. Translocation of extracellular space fluid was observed with a reduction in atrial distension. The secretion of irANP into the atrial lumen occurs less than 15 sec of the reduction in atrial distension. It is therefore suggested that the incremental response of irANP secretion to the reduction in atrial distension is a sequential mechanism of ANP secretion, in which first is the release of ANP from the atrial myocytes into the extracellular space and then second is the translocation of ANP with extracellular space fluid into the atrial lumen with a reduction in atrial distension.

Presence and release of immunoreactive atrial natriuretic peptide in granulosa cells of the pig ovarian follicle

Atrial natriuretic peptide (ANP) has been reported to be locally synthesized in the ovary although its physiological roles are still unknown. To define the origin of ovarian ANP, we demonstrated the presence and release of immunoreactive (ir) ANP in pig granulosa cells and characterized its biochemical properties. Serial dilution curves made with the extracts of pig granulosa cells, their perfusates and follicular fluid were paralleled to the standard curve of ANP. The amount of irANP in the granulosa cell was 2 fg/cell. The total amount of irANP in granulosa cells significantly correlated with the levels of irANP in follicular fluid. Additionally, the total content of irANP in the follicle negatively correlated with the follicular size. On reverse phase HPLC, the major form of irANP in granulosa cells and follicular fluid was high molecular weight but that in perfusate was low molecular weight. In Northern blot analysis, ANP mRNA was detected in the pig granulosa cells. Immunohistochemistry showed ANP prohormone location in granulosa cells of rat ovary. These data strongly suggest that the granulosa cells synthesize and secrete ANP.

Cardiovascular Gap Junction Proteins: Molecular Characterization and Biochemical Regulation

Gap junctions are plasma membrane specializations containing channels which permit the intercellular exchange of ions and small molecules. Gap junction channels are of central importance in electrically excitable tissues such as myocardium where cell-to-cell passage of ions allows propagation of action potentials. Gap junctions are also present in many non-excitable cells (for example endothelial cells) where they may facilitate intercellular exchange of nutrients, metabolites, and signaling molecules as well as ions. The present review will focus on molecular biological and biochemical studies that have enhanced our understanding of the molecular composition of cardiac and vascular gap junction channels and the regulation of the subunit proteins that form them.