Comparison of two muscle fibre staining techniques and their relation to pork quality traits

This study compared two histochemical staining methods of muscle fibres and evaluated their relationship with the meat quality traits of two high-value porcine muscles. Immunohistochemical (IHC) and adenosine triphosphatase (ATPase) staining was used to assess the cross-sectional area and proportion of fibre-types I, IIa, IIx and IIb in the samples of longissimus lumborum (LL) and psoas major (PM) muscles collected one-hour post-mortem from 25 crossbred pigs [Large White_Sire × (Landrace × Large White_Dam)] at an average age of 152 days. Muscles differed in all fibre parameters, except the proportion and relative area of type IIx fibres. The LL muscle exhibited greater fibre cross-sectional areas of all fibre types, higher proportions of type IIb/IIB, and lower proportions of I and IIa fibres than the PM muscle in both staining techniques. These two muscles also differed marginally in moisture, crude protein and intramuscular fat content. The PM muscle showed a low correlation between fibre types and chemical composition, but the LL muscle showed moderate correlations between fibre CSA and area composition for moisture and ash content. After IHC staining, an increase in LL eye muscle area and drip loss were correlated with lower proportions of type I fibres, while a greater proportion of type IIx fibres resulted in increased LL eye muscle area and moisture content. Furthermore, a higher CSA of all fibre types in the LL decreased redness (a*) and moisture content of the muscle. Results showed that IHC is more appropriate than ATPase staining for the assessment of relationships between muscle fibre parameters and meat quality traits in pigs.

Pattern of fibre type distribution within muscle fascicles of pigs (Sus scrofa domestica)

In order to determine breed differences in the fascicle structure of skeletal muscles, longissimus dorsi muscles (LD) and semimembranous muscles (SM), Duroc (D), Large White (LW), German Landrace (GL) and Swedish Landrace (SL) boars were analyzed. Fibre type delineation was based on the method of myofibrillar ATPase staining. The fibres in a fascicle were divided into layers according to their relation to the perimysium. In each layer, the proportions of fibre types I, IIA and IIB were determined and normalized according to the fibre type proportion in the whole fascicle. Generally, a high proportion of IIB fibres on the periphery, a prevalence of type I and IIA in the layer below, and altered proportions of type I, IIA and IIB in layer 3 were found to be the main characteristics of the distribution of fibres in a porcine fascicle. Breed dissimilarity was established in the arrangement of fibre types, particularly in the first and third layer. The breeds that showed the highest proportions of type I and IIB fibres in LD and SM muscles were GL and SL boars.

Effect of early feed restriction on myofibre types and expression of growth-related genes in the gastrocnemius muscle of crossbred broiler chickens

The purpose of this study was to investigate the immediate and long-term effects of early feed restriction (ER) on morphology and gene expression of lateral gastrocnemius muscle. Newly hatched crossbred broiler chickens were allocated into control and ER groups, the latter being free-fed on alternate days from hatch to 14 days of age (14 d), followed by ad libitum feeding as the control group until 63 d. The lateral gastrocnemius muscle was taken at 14 and 63 d, respectively for myofibre typing by both myosin ATPase staining and relative quantification of myosin heavy chain (MyHC) mRNA for slow-twitch (SM), red fast-twitch (FRM) and white fast-twitch (FWM) myofibres. The body weight and lateral gastrocnemius weight were significantly lower in the ER group, accompanied by significantly reduced serum triiodothyronine. The ER group exhibited significantly higher SM and FRM MyHC expression at 14 d, but lower SM expression at 63 d. Myosin ATPase staining revealed a similar pattern. The percentage of SM was higher at 14 d while lower at 63 d in the ER group. These morphological changes were accompanied by changes of mRNA expression for growth-related genes. The ER group expressed lower insulin-like growth factoar I (IGF-I) and higher IGF-I receptor (IGF-IR) at 14 d, yet significantly increased growth hormone receptor and IGF-IR mRNA at 63 d. These results indicate that ER may delay the slow to fast myofibre conversion as an immediate effect, but would result in a lower percentage of slow fibres owing to compensatory growth in the long term, which involves changes of mRNA expression for the growth-related genes in the muscle.

Potassium depletion increases proton pump (H+-ATPase) activity in intercalated cells of cortical collecting duct

Intercalated cells (ICs) from kidney collecting ducts contain proton-transporting ATPases (H+-ATPases) whose plasma membrane expression is regulated under a variety of conditions. It has been shown that net proton secretion occurs in the distal nephron from chronically K+-depleted rats and that upregulation of tubular H+- ATPase is involved in this process. However, regulation of this protein at the level of individual cells has not so far been examined. In the present study, H+-ATPase activity was determined in individually identified ICs from control and chronically K+-depleted rats (9–14 days on a low-K+ diet) by monitoring K+- and Na+-independent H+ extrusion rates after an acute acid load. Split-open rat cortical collecting tubules were loaded with the intracellular pH (pHi) indicator 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and pHiwas determined by using ratiometric fluorescence imaging. The rate of pHi recovery in ICs in response to an acute acid load, a measure of plasma membrane H+-ATPase activity, was increased after K+ depletion to almost three times that of controls. Furthermore, the lag time before the start of pHirecovery after the cells were maximally acidified fell from 93.5 ± 13.7 s in controls to 24.5 ± 2.1 s in K+-depleted rats. In all ICs tested, Na+- and K+-independent pHi recovery was abolished in the presence of bafilomycin (100 nM), an inhibitor of the H+-ATPase. Analysis of the cell-to-cell variability in the rate of pHi recovery reveals a change in the distribution of membrane-bound proton pumps in the IC population of cortical collecting duct from K+-depleted rats. Immunocytochemical analysis of collecting ducts from control and K+-depleted rats showed that K+-depletion increased the number of ICs with tight apical H+ATPase staining and decreased the number of cells with diffuse or basolateral H+-ATPase staining. Taken together, these data indicate that chronic K+ depletion induces a marked increase in plasma membrane H+ATPase activity in individual ICs.

Regulation of Na+, K(+)-ATPase in the rat outer medullary collecting duct during potassium depletion.

Because in outer medullary collecting ducts (OMCD) of K(+)-depleted rats, K+ secretion is abolished, whereas Na+, K(+)-ATPase, which energizes this secretion, is markedly stimulated, it has been proposed that Na+, K(+)-ATPase was mislocated to the apical cell membrane and energized K+ reabsorption. This hypothesis has been supported by paradoxical effects of ouabain in K(+)-depleted compared with normal rats. However, we have recently shown that ouabain inhibits not only Na+, K(+)-ATPase but also apical H+, K(+)-ATPase in the OMCD of K(+)-depleted rats. Therefore, this study was designed to evaluate whether previous observations were accounted for by Na+, K(+)-ATPase or by ouabain-sensitive H+, K(+)-ATPase. Na+, K(+)-ATPase was distinguished from H+, K(+)-ATPase by its insensitivity to Sch-28080. Results indicate that the hydrolytic and transport activities of Na+, K(+)-ATPase, the number of its functional units, and the expression of mRNA of its alpha 1 and beta 1 subunits were increased threefold or more in the OMCD of rats fed a K(+)-depleted diet for 2 wk. By immunofluorescence, Na+, K(+)-ATPase staining was strongly increased in K(+)-depleted rats but remained localized to the basolateral pole of OMCD principal cells. In conclusion, K+ depletion is associated with marked induction of functional Na+, K+ pumps at the basolateral pole of rat OMCD. Therefore, reduced K+ secretion might result from inhibition of apical K+ conductances and stimulation of basolateral K+ recycling. It is proposed that increased Na+, K(+)-ATPase participates in the increased Na+ reabsorption prevailing in collecting ducts of K(+)-depleted rats.

Adaptational changes in renal vacuolar H(+)-ATPase in the rat remnant kidney.

After 7/8 nephrectomy, the remnant kidney exhibits an adaptive increase in acid secretion per nephron. We studied the role of the kidney vacuolar H(+)-ATPase in this adaptational response by anti-H(+)-ATPase immunocytochemistry in kidneys from rats subjected to 7/8 nephrectomy or a sham procedure at 1, 2, and 3 wk after surgery. No changes in H(+)-ATPase staining were apparent in the proximal tubule, thick ascending limb, or distal convoluted tubule. In the cortical collecting ducts, the percentage of intercalated cells with well-polarized apical and well-polarized basolateral H(+)-ATPase staining was significantly higher in the remnant kidneys at 1, 2, and 3 wk. In the medullary intercalated cells of the sham-operated kidneys, relatively few cells had predominant plasma membrane H(+)-ATPase staining. In the remnant kidneys, the percentage of medullary intercalated cells with predominant plasma membrane H(+)-ATPase staining was increased significantly at 1 and 2 wk after surgery, returning to sham-operated values at 3 wk. The results indicate that intercalated cells in the collecting ducts of both cortex and medulla participate in the adaptation to reduction in renal mass by changing the steady-state distribution of H(+)-ATPase, similar to changes that occur with chronic acid administration.

Colocalization of H-ATPase and H,K-ATPase immunoreactivity in the rat kidney.

Urinary acidification in the collecting duct (CD) is via V-type H-ATPase and P-type H,K-ATPase. The localization and polar distribution of H-ATPase in intercalated cells (IC) have been well studied. The localization of H,K-ATPase to IC has been reported, but its intracellular distribution has not been defined. To colocalize these pumps, a murine monoclonal antibody (E11) to the 31-kd subunit of the H-ATPase and a rabbit antiserum (HK alpha N2) to a synthetic peptide based on the N terminus of the hog gastric H,K-ATPase alpha-subunit were used. In immunocytochemical staining of rat kidney, H,K-ATPase was present only in the IC of the CD with the same polar distribution as H-ATPase. The preabsorption of HK alpha N2 with affinity-purified bovine H-ATPase did not affect the H,K-ATPase staining, whereas preabsorption with the immunizing synthetic peptide eliminated immunoreactivity. HK alpha N2 stained only parietal cells in the rat gastric mucosa, whereas preimmune serum and E11 showed no immunoreactivity. On immunoblots of rat gastric mucosal microsomes, HK alpha N2 labeled a single 94-kd band, and this staining disappeared after preabsorption with the immunizing synthetic peptide. HK alpha N2 labeled no bands on immunoblots of affinity-purified bovine H-ATPase. All immunochemistry was negative with preimmune serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Enrichment of rab11, a small GTP-binding protein, in gastric parietal cells

Parietal cell secretion of acid requires the coordinated fusion of H(+)-K(+)-adenosinetriphosphatase (ATPase)-containing tubulovesicles with a secretory canalicular target membrane. We have previously reported the presence of rab2 on parietal cell tubulovesicles (L. H. Tang, S. A. Stoch, I. M. Modlin, and J. R. Goldenring. Biochem. J. 285: 715-719, 1992). Since 60% of the small GTP-binding protein sequences obtained from parietal cells were > 95% homologous with human rab11 (J. R. Goldenring, K. R. Shen, H. D. Vaughan, and I.M. Modlin. J. Biol. Chem. 268: 18419-18422, 1993), we sought to study rab11 in gastric parietal cells. A complete rab11 sequence was obtained, and the deduced amino acid sequence of rabbit rab11 was identical to that for human. Rab11 mRNA was present throughout the gastrointestinal mucosa. mRNA for both rab11 and rab2 were enriched in isolated parietal cells compared with chief cells. A polyclonal antiserum against rab11 labeled a single 25-kDa band in isolated parietal cells. Immunostaining of rat fundic tissue demonstrated prominent staining of parietal cells. Rab11 staining cosegregated with alpha-H(+)-K(+)-ATPase staining in enriched preparations of rabbit parietal cell tubulovesicles. These results suggest that rab11 is enriched in parietal cells and is associated with intracellular tubulovesicles.

Cellular distribution of H(+)-ATPase following acute unilateral ureteral obstruction in rats

Unilateral ureteral obstruction for 24 h produces an acidification defect in the rat kidney that closely resembles the human disorder. We examined the role of renal vacuolar H(+)-ATPase distribution and content in the generation of the postobstructive abnormality in distal hydrogen ion secretion. Rats were subjected to unilateral ureteral obstruction for 24 h, and the obstructed and contralateral kidneys were removed at 3 h, 5 days, and 10 days after release of the obstruction. The postobstructed and contralateral kidneys and kidneys from sham-operated rats were analyzed for intercalated cell number and subtype and for the cellular distribution of ATPase staining by means of a monoclonal antibody specific for the 31-kDa subunit of the vacuolar H(+)-ATPase. No change in the number or distribution of subtypes was detected in the cortex nor in the outer or inner stripe of the outer medulla. Immunoreactive H(+)-ATPase increased in both the cortex and medulla at 3 h after obstruction, and thereafter it declined to control values. The major morphological changes in H(+)-ATPase staining detected were an alteration in the intracellular distribution of the enzyme, which we refer to as discontinuity of (or “gaps” in) apical staining, and a decrease in the percent of intercalated cells showing a rim (or plasma membrane) staining pattern in the inner medulla. The changes observed may be a morphological representation of the physiological abnormalities underlying the postobstructive acidification defect.