Calpain activation and proteolysis in postmortem goose muscles.

Meat tenderness is considered as the most important criterion for meat quality by consumers and can be improved by the actions of endogenous proteases, mainly calpains, during postmortem storage at 0-5°C. The purpose of this study, therefore, was to examine the postmortem calpain activation and proteolysis in breast (BM) and leg and thigh (LM) muscles of White Roman goose. BM and LM were taken from goose carcasses (n = 15) at 0 (10-15 min postmortem), 1, 3, and 7 days of storage at 5°C. The decrease in postmortem pH, calpain-1 and -11 activities, and contents of the calpain-1 80 kDa subunit and desmin was more rapid (p < .05) in BM than in LM. Our results show that postmortem proteolysis was more extensive in BM than in LM of White Roman goose, not only because the difference in fiber type composition between two muscles, but because the rate and extent of calpain activation were greater in BM as well. These results may provide useful information to optimize meat processing for different muscles in goose industry.

Postmortem role of calpain in Chinese and Wuzong goose muscles.

The purpose of this study was to compare postmortem proteolysis and tenderization between Chinese and Wuzong goose breast muscles. Four months old Chinese (CG, n = 15) and Wuzong (WZ, n = 15) goose carcasses were vacuum-packaged 10 to 15 min postmortem and stored at 5°C. Breast (Pectoralis major) samples from each carcass were sampled at 0 (∼10 min postmortem), 1, 3, and 7 D of storage. Our results showed that the decrease in pH and calpain-1 activity was not different in CG and WG samples. However, the decrease in calpain-11 activity, desmin content, and shear force were more rapid (P < 0.05) in WZ than in CG samples. Our results indicate that postmortem proteolysis and tenderization of goose breast muscle were more extensive in WZ than in CG goose muscle.

Effect of age on calpain changes in postmortem goose muscle.

Meat tenderization can be affected by a variety of factors including animal age. However, results obtained from goose, chicken, turkey, and ostrich studies have shown that the effects of age on meat tenderization were insignificant, which may be due to a very limited age difference in birds used in those studies. Therefore, the purpose of this study was to investigate the effects of animal age on postmortem proteolysis and tenderization of breast muscle from developing and mature White Roman geese. Goose carcasses from mature (50 mo old, n = 10) and young (3 mo old, n = 10) geese were vacuum-packaged and stored at 5°C within 10 min postmortem. Breast (pectoralis major) samples were taken at 0 (∼10 min postmortem), 1, 3, and 7 D of storage. Our results showed that the decrease in pH, calpain-1 and -11 activities, desmin content and shear force, as well as the increase in myofibrillar fragmentation index were more rapid (P < 0.05) in young than in mature goose breast. These results suggest that postmortem proteolysis and tenderization of goose muscle are extensively affected by age.

µ-Calpain is involved in the postmortem proteolysis of gizzard smooth muscle.

Postmortem changes in proteins that have been implicated in affecting muscle integrity were examined in goose (GG) and duck (DG) gizzard smooth muscle stored at 5°C. GG and DG smooth muscles were sampled at 0, 1, 3 and 7 day of storage. The pH was approximately 7 in both GG and DG samples during postmortem storage. Casein zymograms showed that 0-day µ-calpain activity was higher (p<0.05) in GG than in DG samples. As postmortem time progressed, µ-calpain was activated and autolyzed more extensively in GG than in DG samples. However, µ/m-calpain remained relatively stable in both samples. Western blots indicated that postmortem desmin degradation was more rapid in GG than in DG samples. In contrast, α-actinin remained nearly unchanged in both samples. Therefore, our results suggest that µ-calpain has an important role in the postmortem proteolysis of gizzard smooth muscle.

Immunocytochemical distribution of somatotrophs in porcine anterior pituitary.

The objective of this immunohistochemical study was to identify the spatial distribution patterns of growth hormone (GH) secreting cells (somatotrophs) in the newborn and prepubertal porcine pituitary. No differences were observed among the total somatotrophs per unit area across the three ages. There were, however, changes in spatial distribution of somatotrophs in porcine pituitary with developmental age. Distinctive characteristics of the pattern included a high population of somatotrophs (44 +/- 1.2; mean +/- standard error of the mean per 30,495 microm(2)) in regions 1 and 5 and a low population (22 +/- 1.4) in regions 2 and 4 at each level (P < 0.05). Somatotrophs increased 55% in region 3 from proximal to distal levels at all ages. With increasing age, however, somatotrophs in region 3 at the proximal level decreased 33%. From these results, we suggest that there may be regional specificity of cellular differentiation and transformation to facilitate GH secretion to meet the need for endocrine regulation as the animal ages.

Number of secretory vesicles in growth hormone cells of the pituitary remains unchanged after secretion.

Immunogold-labeled transmission electron microscopy (TEM) was used to determine the total number of secretory vesicles in resting and in growth hormone (GH)-stimulated porcine pituitary cells. We identified three categories of vesicles: filled, empty, and partly empty. Resting GH cells contained more than twice as many filled vesicles than did the stimulated ones. Stimulated cells, however, contained nearly twice as many empty vesicles and 2.5 times more partly empty vesicles than did resting cells. Secretory vesicles in GH cells further revealed the localization of GH only in electron-dense vesicles in both resting and stimulated cells. The total number of secretory vesicles did not change after secretion. These results are consistent with a mechanism that, after stimulation of secretion, vesicles transiently dock and fuse at the fusion pore to release vesicular contents.

Reconstituted fusion pore.

Fusion pores or porosomes are basket-like structures at the cell plasma membrane, at the base of which, membrane-bound secretory vesicles dock and fuse to release vesicular contents. Earlier studies using atomic force microscopy (AFM) demonstrated the presence of fusion pores at the cell plasma membrane in a number of live secretory cells, revealing their morphology and dynamics at nm resolution and in real time. ImmunoAFM studies demonstrated the release of vesicular contents through the pores. Transmission electron microscopy (TEM) further confirmed the presence of fusion pores, and immunoAFM, and immunochemical studies demonstrated t-SNAREs to localize at the base of the fusion pore. In the present study, the morphology, function, and composition of the immunoisolated fusion pore was investigated. TEM studies reveal in further detail the structure of the fusion pore. Immunoblot analysis of the immunoisolated fusion pore reveals the presence of several isoforms of the proteins, identified earlier in addition to the association of chloride channels. TEM and AFM micrographs of the immunoisolated fusion pore complex were superimposable, revealing its detail structure. Fusion pore reconstituted into liposomes and examined by TEM, revealed a cup-shaped basket-like morphology, and were functional, as demonstrated by their ability to fuse with isolated secretory vesicles.

Structure and composition of the fusion pore.

Earlier studies using atomic force microscopy (AFM) demonstrated the presence of fusion pores at the cell plasma membrane in a number of live secretory cells, revealing their morphology and dynamics at nm resolution and in real time. Fusion pores were stable structures at the cell plasma membrane where secretory vesicles dock and fuse to release vesicular contents. In the present study, transmission electron microscopy confirms the presence of fusion pores and reveals their detailed structure and association with membrane-bound secretory vesicles in pancreatic acinar cells. Immunochemical studies demonstrated that t-SNAREs, NSF, actin, vimentin, alpha-fodrin and the calcium channels alpha1c and beta3 are associated with the fusion complex. The localization and possible arrangement of SNAREs at the fusion pore are further demonstrated from combined AFM, immunoAFM, and electrophysiological measurements. These studies reveal the fusion pore or porosome to be a cup-shaped lipoprotein structure, the base of which has t-SNAREs and allows for docking and release of secretory products from membrane-bound vesicles.

Use of actin isoform-specific antibodies to probe the domain structure in three smooth muscles.

We investigated the location of actin isoforms in relation to each other and to filament attachment sites by studying the edge-to-edge distribution of both immunofluorescence and immunogold probes in smooth muscle cells from three sources. Antibodies to alpha- or alpha,gamma-actin labeled uniformly across smooth muscle cells from each source. Antibodies to beta-cytoplasmic actin were concentrated on and near dense bodies, especially in gizzard smooth muscle, but were also located throughout the filament compartment. Double immunofluorescent labeling with antibodies to alpha- or alpha/gamma- and to beta-actin shows overlap of label at dense bodies and attachment plaques. Double immunofluorescent labeling with antibodies to alpha-actinin and to beta-actin identified dense bodies and attachment plaques as sites of colocalization. Immunogold labeling with anti-desmin was most prominent near dense bodies in the gizzard and was widely dispersed in vas deferens and arterial smooth muscle cells. Our results indicate that there is extensive overlap between the locations of contractile and cytoskeletal elements and, thus, do not support the two-domain model of smooth muscle structure. Tissue-specific organizational motif differences were seen when gizzard, vas deferens, and artery were compared and suggest that one model may not apply to these three smooth muscles.

G(alpha)(i3) in pancreatic zymogen granules participates in vesicular fusion.

Earlier studies indicated that a G(i)-like protein localized in pancreatic zymogen granule (ZG) membrane mediates vesicle swelling, and is a potentially important prerequisite for vesicle fusion at the cell plasma membrane (PM) [Jena et al. (1997) Proc. Natl. Acad. Sci. USA 94, 13317-13322]. In the present study, we demonstrate the presence of G(alpha)(i3) immunoreactivity in ZGs of rat exocrine pancreas using immunoblot assays, light and electron immunomicroscopy. Since GTP has been implicated in the fusion of isolated ZG with PM fractions [Nadin et al. (1989) J. Cell Biol. 109, 2801-2808], the potential role of ZG-associated G(alpha)(i3) was investigated. Immunoblot assays demonstrate an increase in G(alpha)(i3) protein in ZGs isolated from carbamylcholine stimulated pancreas. Thin layer chromatography shows an increase in GTP hydrolysis by GTPase in ZGs isolated from stimulated compared to resting pancreas. In vitro fusion assays demonstrate that ZGs isolated from carbamylcholine-stimulated pancreatic lobules fuse with the PM at a greater potency in the presence of GTP, mastoparan (G protein agonist) and its analogue mas7. Furthermore, G(alpha)(i3)-specific a recombinant GAIP (G alpha interacting protein), potentiates ZG-PM fusion in the presence of GTP but not in presence of the non-hydrolyzable GTP analogue Gpp(NH)p. Our immunoblot analysis demonstrates the recruitment of Galpha(i3) immunoreactivity to ZG from stimulated acinar cells, and these isolated ZGs are more potent and efficient in fusing with plasma membrane fractions, suggesting the possible involvement of G(alpha)(i3) in ZG-PM fusion. The participation of ZG-associated G(alpha)(i3) in ZG-PM fusion is further confirmed by the influence of the G(alpha)(i3)-specific GAIP, which is known to interact specifically with G(alpha)(i3), and not with G(alpha)(i2) or G(alpha)(q) [DeVries et al. (1995) Proc. Natl. Acad. Sci. USA 92, 11916-11920]. Additionally, our data suggest that GTP hydrolysis is a requirement for ZG-PM fusion since GAIP in the presence of Gpp(NH)p shows little or no effect on fusion, whereas GAIP in the presence of GTP significantly potentiates ZG-PM fusion. Our studies suggest a possible role for ZG-associated G(alpha)(i3) in ZG-PM fusion.

Aquaporin 1 regulates GTP-induced rapid gating of water in secretory vesicles.

The swelling of secretory vesicles has been implicated in exocytosis, but the underlying mechanism of vesicle swelling remains largely unknown. Zymogen granules (ZGs), the membrane-bound secretory vesicles in exocrine pancreas, swell in response to GTP mediated by a G(alpha)i3 protein. Evidence is presented here that the water channel aquaporin-1 (AQP1) is present in the ZG membrane and participates in rapid GTP-induced vesicular water gating and swelling. Isolated ZGs exhibit low basal water permeability. However, exposure of granules to GTP results in a marked potentiation of water entry. Treatment of ZGs with the known water channel inhibitor Hg2+ is accompanied by a reversible loss in both the basal and GTP-stimulatable water entry and vesicle swelling. Introduction of AQP1-specific antibody raised against the carboxyl-terminal domain of AQP1 blocks GTP-stimulable swelling of vesicles. Our results demonstrate that AQP1 associated at the ZG membrane is involved in basal as well as GTP-induced rapid gating of water in ZGs of the exocrine pancreas.

Structure and dynamics of the fusion pore in live cells.

Atomic force microscopy reveal pit-like structures typically containing three or four, approximately 150 nm in diameter depressions at the apical plasma membrane in live pancreatic acinar cells. Stimulation of secretion causes these depressions to dilate and return to their resting size following completion of the process. Exposure of acinar cells to cytochalasin B results in decreased depression size and a loss in stimulable secretion. It is hypothesized that depressions are the fusion pores, where membrane-bound secretory vesicles dock and fuse to release vesicular contents. Zymogen granules, the membrane-bound secretory vesicles in exocrine pancreas, contain the starch digesting enzyme, amylase. Using amylase-specific immunogold labeling, localization of amylase at depressions following stimulation of secretion is demonstrated. This study confirms depressions to be the fusion pores in pancreatic acinar cells. High-resolution images of the fusion pore in live pancreatic acinar cells reveal the structure in much greater detail than has previously been observed.