Thomas A. Heming

6 Yolk Absorption in Embryonic and Larval Fishes

Publisher Summary This chapter discusses the yolk absorption in embryonic and larval fishes. The rate and efficiency of yolk absorption are influenced by a number of environmental factors, including temperature, light, oxygen concentration, and salinity. Fish eggs are not motile, and thus developing embryos are unable to actively exploit the most favorable environments available, at least until after hatching. In teleost eggs, the yolk syncytium together with overlaying mesoderm and ectoderm spreads to enclose the entire yolk mass. Endoderm does not follow the movement of the teleost blastodisc rim and, consequently, the yolk is not enclosed by an endodermal layer. Yolk platelets are mobilized more rapidly than the oil globule from the yolk mass, especially after hatching. The rate at which yolk reserves are depleted must be a function of the surface area of the absorptive layer and the metabolic activity of that layer.

Bactericidal activity of alveolar macrophages is suppressed by V-ATPase inhibition

Bafilomycin A1, a selective inhibitor of V-type H+-translocating ATPase (V-ATPase), may be a useful adjunct in cancer chemotherapy (Altan et al. [1998] J Exp Med 187:1583–1598). Therapeutic uses of the enzyme inhibitor need to consider the agents potential effects on normal (nontumor) cells. This study determined the effects of bafilomycin A1 on resident alveolar macrophages (mφ). Treatment of alveolar mφ with bafilomycin A1 (10 μM, 1 h) caused a significant decrement in cytosolic pH. This was accompanied by marked alteration of mφ bactericidal capabilities. The enzyme inhibitor caused a marginal reduction in the phagocytosis of opsonized Staphylococcus aureus and significantly suppressed intracellular killing of the phagocytosed bacteria. In keeping with the effects on intracellular killing, bafilomycin A1 significantly reduced the production of reactive oxygen species (ROS). On the other hand, cell spreading was enhanced significantly by bafilomycin A1. Comparable changes in ROS generation and mφ spreading were produced by altering cytosolic pH through changes in extracellular pH (pHo) in the absence of bafilomycin A1. These findings suggest that the agents effects on ROS production and mφ spreading were related to the accompanying changes in cytosolic pH. The enzyme inhibitor also altered mφ morphology, leading to the shortening of microvilli and focal loss of surface ruffles. These morphologic effects differed from those produced by altering cytosolic pH by changes in pHo. The results demonstrate that V-ATPase activity is an important determinant of mφ functioning and structure. Therapeutic use of V-ATPase inhibitors might be expected to compromise the bactericidal activity of alveolar mφ.

Effects of bafilomycin A1 on functional capabilities of LPS-activated alveolar macrophages.

Resident alveolar macrophages (mφ) possess plasmalemmal vacuolar‐type H+‐ATPase (V‐ATPase) that plays a crucial role in regulation of intracellular pH (pHi). To assess the importance of this V‐ATPase to mφ effector functions, resident alveolar mφ from rabbits were activated with E. coli‐derived lipopolysaccharide (LPS) and exposed to bafilomycin A1 a specific inhibitor of V‐ATPase. Bafilomycin caused a significant cytosolic acidification in both the absence and presence of CO2‐HCO3 ‐, and in both unstimulated and activated mφ. Superoxide production and Fc receptor‐mediated phagocytosis also were reduced in bafilomycin‐treated mφ. Similar effects were elicited by acidifying the cytoplasm in the absence of bafilomycin, by lowering extracellular pH (pHo) from 7.4 to 6.5–6.6. Thus, the effects of bafilomycin on phagocytosis and superoxide production probably were related to cytosolic acidification, secondary to blockade of V‐ATPase‐mediated H+ extrusion across the plasma membrane. Conversely, bafilomycin significantly increased TNF‐α release. This effect cannot be explained by a bafilomycin‐induced acidosis because acidic pHo significantly reduced TNF‐α release. The results demonstrate that V‐ATPase activity is an important determinant of the effector functions of LPS‐activated mφ. J. Leukoc. Biol. 57: 275–281; 1995.

Effects of extracellular pH on tumour necrosis factor-α production by resident alveolar macrophages

Cellular acid-base status has been found to exert selective actions on the effector functions of activated macrophages (mphi). We examined the effects of extracellular pH (pH(o)) on the production of tumour necrosis factor-alpha (TNF-alpha) induced by lipopolysaccharide (LPS) in resident alveolar mphi. Cells were obtained by bronchoalveolar lavage of rabbits, activated in vitro with LPS, and cultured at pH(o) 5.5, 6.5 or 7.4 for up to 18 h. The relative abundance of TNF-alpha mRNA peaked at approximately 2 h. The peak transcript abundance was increased at lower pH(o) values. This finding probably reflected pre-transcription/transcription effects of pH, in as much as the stability of TNF-alpha mRNA induced with phorbol ester was unaffected by the experimental pH(o) values. TNF-alpha secretion by LPS-treated mphi decreased at lower pH(o) values. The TNF-alpha content of mphi-conditioned media decreased progressively with decrements in pH(o). The reduced TNF-alpha secretion at pH(o) 5.5 was accompanied by an increase in the cytosolic TNF-alpha content (compared with that at pH(o) 7.4), indicating that pH(o) altered TNF-alpha secretion due, in part, to the intracellular retention of synthesized cytokine (i.e. a post-translation effect). The data show that pH(o) has multiple effects (pre-transcription/transcription and post-translation) on TNF-alpha production induced by LPS in resident alveolar mphi. These results suggest that the role of alveolar mphi in inflammatory responses is modulated by pH(o), which may be important in tumours/abscesses and sites of infection where the external milieu is acidic.

3 - Carbonic Anhydrase and Respiratory Gas Exchange

This chapter discusses the carbonic anhydrase (CA) and respiratory gas exchange in fishes. The enzyme, carbonic anhydrase, catalyzes the reversible hydration/ dehydration of carbon dioxide. It is found that from the perspective of respiratory gas exchange in fish, CA is found in tissues at every major step in the process from actively metabolizing tissue (e.g., muscle), where CO 2 is produced, to elements in the circulatory system, where CO 2 transport takes place, and to the gills, where CO 2 is excreted. In the systemic circulation of elamobranches and teleosts, mass action favors hydration and product inhibition is minimized through the buffering of protons by the high concentrations of hemoglobin within the red cell and by the removal of HCO 3 - through the chloride shift. It has been suggested that CA, associated with the extracellular surface of the sarcolemma, facilitates NH 3 transport through the generation of protons through the catalyzed hydration of CO 2 and these results in ammonia trapping as NH 4 + in the extracellular fluid boundary layer, thus, maintaining the trans-sarcolemma PNH 3 gradient.

RESPIRATORY, ACID-BASE AND IONIC STATUS OF KEMP'S RIDLEY SEA TURTLES (LEPIDOCHELYS KEMPI) SUBJECTED TO TRAWLING

Abstract 1. Kemps ridley sea turtles were placed in a commercial shrimp trawl equipped with a turtle excluder device (TED), resulting in a burst of apneic swimming and a brief bout of forced submergence (⩽7.3 min). 2. Trawl tests induced a significant non-respiratory (metabolic) acidosis; blood pH declined almost 0.4 units and plasma [lactate] increased 6-fold from pre- to post-trawl conditions. 3. Significant changes in blood parameters occurred regardless of the duration of forced submergence (range 2.7–7.3 min), suggesting apneic activity was a significant contributor to the observed acid-base imbalance.

Post-transcriptional effects of extracellular pH on tumour necrosis factor-α production in RAW 246.7 and J774 A.1 cells

The present studies determined the effects of extracellular pH (pH(o)) on the production of tumour necrosis factor-alpha (TNF-alpha) in the macrophage-like cell lines RAW 246.7 and J774 A.1. The cells were activated with lipopolysaccharide (LPS) at pH(o) 5.5, 6.5 or 7.4. TNF-alpha gene transcription was monitored by Northern blot analysis. Synthesis of the cytokine was monitored by ELISA measurements of the TNF-alpha content of cell-conditioned media (extracellularly released TNF-alpha) and cell lysates (cytosolic TNF-alpha). The magnitude of the TNF-alpha response differed markedly between the two cell lines. RAW cells were more responsive to LPS than were J774 cells. However, the effects of pH(o) on TNF-alpha production were similar in the two cell lines. TNF-alpha gene transcription was insensitive to experimental pH(o). The pH(o) had no effect on the abundance of TNF-alpha mRNA at 2, 4 or 18 h. Nonetheless, synthesis of TNF-alpha was affected significantly by pH(o). The TNF-alpha contents of cell-conditioned medium and cell lysate at 18 h were reduced progressively at lower pH(o) values. The data indicate that pH(o) alters TNF-alpha production in RAW and J774 cells at a post-transcriptional level. These findings suggest that pH(o) influences the phenotypic responses of macrophages to activating stimuli and modifies the role that macrophages play in inflammatory and immune actions.

Na+‐H+ exchange in resident alveolar macrophages: activation by osmotic cell shrinkage

Intracellular pH (pH;) homeostasis in resident alveolar macrophages (mφ) under nominally CO2‐free conditions is mediated primarily by the activity of plasmalemmal H+‐ATPase, The mφ also possess an Na+‐H+ exchanger (NHE) but this mechanism has no de tectable role in pHi regulation in the physiologic range. To further explore the physiological significance of the NHE in this cell type, resident alveolar mφ from rabbits were subjected to a hyperosmotic challenge (620 mOsm/kg) in the nominal absence of C02‐HCO3 ‐. Os motic cell shrinkage was accompanied by an amiloridesensitive increase in baseline pHi The NHE‐mediated rate of pH; recovery from intracellular acid loads also in creased under hyperosmotic conditions. Cell shrinkage caused an alkaline shift in the pHi set point of the NHE without altering the exchangers affinity for extracellular Na+. The results indicate that Na+‐H+ exchange in resi dent alveolar mφ is activated by osmotic cell shrinkage and imply that the NHE may be involved in volume regulatory responses of the cell. J. Leukoc. Biol. 57: 609–616; 1995.

Effects of lidocaine on cytosolic pH regulation and stimulus-induced effector functions in alveolar macrophages.

Abstract. Local anesthetics influence a variety of stimulus-induced effector functions in leukocytes. The present study determined the effects of lidocaine on intracellular pH (pHi) regulation, superoxide production, and tumor necrosis factor-α (TNF-α) release in alveolar macrophages (mφ). Resident mφ were obtained by bronchoalveolar lavage of rabbits. The cells were subjected to an intracellular acid load, and subsequent pHi recovery was followed in the presence or absence of bafilomycin A1, a specific inhibitor of V-type H+-translocating ATPase (V-ATPase) or amiloride, an inhibitor of Na+/H+ exchange. Lidocaine slowed pHi recovery in a dose-dependent manner. Pretreatment (1 h) with 2.5 mM lidocaine abolished Na+/H+ exchange and reduced the V-ATPase-mediated component of pHi recovery. Lidocaine also significantly depressed the superoxide production induced by phorbol ester. TNF-α release induced by endotoxin was not affected significantly by the local anesthetic. Macrophage viability (trypan blue exclusion) and cellular ATP concentration were unaffected. These results indicate that lidocaine inhibits pHi regulatory mechanisms in alveolar mφ. This disruption of pHi regulation could contribute to inhibitory actions of lidocaine on mφ effector functions.

Effects of myristate phorbol ester on V-ATPase activity and Na(+)-H+ exchange in alveolar macrophages.

The roles of protein kinase G (PKC) in regulation of the plasmalemmal vacuolar‐type H+‐ATPase (V‐ATPase) and Na+‐H+ exchanger (NHE) of rabbit alveolar macro phages (mφ) were investigated using phorbol 12‐myristate 13‐acetate (PMA). At an extracellular pH (pHo) of 7.4 (nominal absence of CO2‐HCO‐ 3), PMA caused a dose‐dependent increase in the rate of cellular H+ extrusion with little change in intracellular pH (pHi). PMA caused a prolonged cytosolic acidification at pHo < 6.8. PMA‐induced changes in pHi were sensitive to bafilomycin A1, but were insensitive to amiloride. Studies of pHi recovery following intracellular acid challenge showed that both V‐ATPase and the NHE were up‐regulated by PMA. An inactive analog, 4α‐phorbol, had no detectable effects on pHi homeostasis. These data indicate that (a) PKC is involved in regulation of V‐ATPase and the NHE of resident alveolar mφ and (b) V‐ATPase is the predominant mechanism for pHi homeostasis in unstimulated and PMA‐activated mφ. J. Leukoc. Biol. 57: 600–608; 1995.