Nerve-induced secretion of parotid acinar granules in cats

Summary Electron microscopy of cat parotid glands revealed great heterogeneity in the secretory granules of normal unstimulated acinar cells. Electrical stimulation of the parasympathetic nerve to the gland evoked a copious flow of parotid saliva which was accompanied by an extensive depletion of the secretory granules from the acinar cells. Exocytosis was captured as it was occurring by means of perfusion-fixation, and showed that the events occur in a conventional manner. Stimulation of the sympathetic nerve caused only a very small flow of saliva, and no acinar degranulation was detected. It can be concluded that the parasympathetic secretomotor axons provide the main drive for parotid acinar degranulation in the cat. This contrasts with the rat in which sympathetic impulses provide the main stimulus for parotid acinar degranulation. These dissimilarities serve to emphasise how extensively species differences may influence autonomic responses in salivary glands.     

Hydrocortisone regulates arylsulfatase A (cerebroside-3-sulfate-3-sulfohydrolase) by decreasing the quantity of the enzyme in cultures of cells dissociated from embryonic mouse cerebra

Previous work from our laboratory (Biochem. J. 219:689–697 (1984) had shown that hydrocortisone stimulated the net accumulation of the myelin-specific sulfolipid in cultures of cells dissociated from embryonic mouse cerebra. This accumulation caused by hydrocortisone was shown to be due to a decrease of sulfolipid degradation by arylsulfatase A (ASA) and not due to a stimulation of its synthesis by a sulfotransferase. Both ASA activity and the turnover of sulfolipid were decreased by hydrocortisone to 60–62% of untreated cells. In current work the same decrease in enzyme activity was obtained and enzyme linked immunosorbent assays demonstrate that hydrocortisone decreased the number of ASA protein molecules to 61% of untreated cells [(-)hydrocorcortisone 0.31±0.06 ng ASA/g protein; (+)hydrocortisone: 0.18±0.04 ng ASA/g protein]. This decrease in the number of ASA molecules correlates well with the decrease in both the enzyme activity and the sulfolipid turnover, which suggests that the major mode of inhibition of ASA activity by hydrocortisone involves a decrease in the concentration of ASA in the cells rather than some other mechanism of inhibition.The material in this paper has been included in a dissertation submitted by A.J.M. in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Temple University.     

Maturation-related changes in mass and elemental contents of secretory granules as measured by electron-microprobe

Summary The relationship between granule density, protein content, and Ca and S contents were studied in two secretory granule fractions, from parotid glands of the rat, previously shown to constitute different stages in granule maturation. The density of the lighter fraction was between 1.133 and 1.142 g/ml, while that of the heavier fraction was greater than 1.142 g/ml. The mean protein content of the denser granules was 12% greater than that of the lighter granules (P<0.03), while the dry-mass elemental concentrations in the two granule fractions were unchanged. These results indicate that protein is added to granules during the maturation process (presumably by vesicular traffic), and that the resulting increase in granule density is not driven simply by decrease in water content and/or increased concentrations of inorganic Ca or S in the granules. The elemental concentration values also indicate that the diffusible elements permeate the granule membrane during the fractionation procedures.     

Effect of calf removal on serum luteinizing hormone and cortisol concentrations in postpartum beef cows

Serum luteinizing hormone (LH) and cortisol concentrations were measured in ten fall calving, Angus cows averaging 38 +/- 8 days postpartum. Calves from five cows were weaned at the beginning of the study. Blood samples were collected at 20 min. intervals for 48 h after weaning and for 8 h on day 4 and day 6 postweaning. Mean serum LH concentrations increased (P<0.01) in weaned cows (W) from 0.55 +/- 0.01 ng/ml at time of calf removal to 1.3 +/- 0.04 ng/ml 48 h afterwards. Comparable LH concentrations for suckled cows (S) were 0.65 +/- 0.08 ng/ml and 0.62 +/- 0.03 ng/ml respectively. Average serum LH concentrations at 48 h after weaning were greater (P<0.01) for W cows than S cows and a treatment by time interaction occurred (P<0.01) with serum LH concentrations increasing (P<0.01) from time of calf removal to 48 h after calf removal in W cows. Frequency of LH peaks increased (P<0.01) in W cows and by 48 h after weaning was greater (P<0.01) in W cows than in S cows. Magnitude of LH peaks did not differ between the two groups. Serum cortisol concentrations were not different between W and S cows except for a transient elevation (P<0.01) in W cows from 7.6 +/- 0.9 ng/ml to 11.9 +/- 1.0 ng/ml 9 to 12 h after calf removal. Since serum LH concentrations were increased in W cows but not in S cows at 48 h and serum cortisol concentrations increased transiently in W cows we suggest that circulating cortisol levels may not be a physiological inhibitor of LH secretion in the suckled postpartum beef cow.     

Structural implications of primary sequences from a family of Balbiani ring-encoded proteins inChironomus

Summary DNA sequencing has revealed an internal, tandemly repetitive structure in the family of giant polypeptides encoded by three types of Balbiani ring (BR) genes, in three different species ofChironomus. Each major BR repeat can be subdivided into two halves: a region consisting of short subrepeats and a more constant region that lacks obvious subrepeats. Comparative predictions of secondary structure indicate that an -helical segment is consistently present in the amino-terminal half of the constant region in all known BR proteins. Comparative predictions, coupled with consideration of the known phosphorylation of serine and threonine residues in BR proteins, suggest that the -helical structure may also extend into the carboxy-terminal half of the constant region, possibly interrupted by -turn(s). However, it is also possible that the structure is variable, and that a -strand is present in that half in some cases. All of the constant regions conserve one methionine and one phenylalanine residue, as well as all four cysteines; these residues presumably play roles in the packing or cross-linking of aligned constant regions. The structure of the subrepeat region is not clear, but the prevalence of a tripeptide pattern (basic-proline-acidic) suggests some type of structural regularity, possibly an extended helix. The possible significance of these conserved molecular features is discussed in the context of how they may serve the elasticity, insolubility, and hydrophilicity of the fibrils and threads formed by the BR polypeptides.     

A Bicarbonate- and Weak Acid-permeable Chloride Conductance Controlled by Cytosolic Ca2+ and ATP in Rat Submandibular Acinar Cells

A Ca²⁺-activated Cl⁻ conductance in rat submandibular acinar cells was identified and characterized using whole-cell patch-clamp technique. When the cells were dialyzed with Cs-glutamate-rich pipette solutions containing 2 mm ATP and 1 μm free Ca²⁺ and bathed in N-methyl-d-glucamine chloride (NMDG-Cl) or Choline-Cl-rich solutions, they mainly exhibited slowly activating currents. Dialysis of the cells with pipette solutions containing 300 nm or less than 1 nm free Ca²⁺ strongly reduced the Cl⁻ currents, indicating the currents were Ca²⁺-dependent. Relaxation analysis of the ``on' currents of slowly activating currents suggested that the channels were voltage-dependent. The anion permeability sequence of the Cl⁻ channels was: NO⁻ ₃ (2.00) > I⁻ (1.85) ≥ Br⁻ (1.69) > Cl⁻ (1.00) > bicarbonate (0.77) ≥ acetate (0.70) > propionate (0.41) ≫ glutamate (0.09). When the ATP concentration in the pipette solutions was increased from 0 to 10 mm, the Ca²⁺-dependency of the Cl⁻ current amplitude shifted to lower free Ca²⁺ concentrations by about two orders of magnitude. Cells dialyzed with a pipette solution (pCa = 6) containing ATP-γS (2 mm) exhibited currents of similar magnitude to those observed with the solution containing ATP (2 mm). The addition of the calmodulin inhibitors trifluoperazine (100 μm) or calmidazolium (25 μm) to the bath solution and the inclusion of KN-62 (1 μm), a specific inhibitor of calmodulin kinase, or staurosporin (10 nm), an inhibitor of protein kinase C to the pipette solution had little, if any, effect on the Ca²⁺-activated Cl⁻ currents. This suggests that Ca²⁺/Calmodulin or calmodulin kinase II and protein kinase C are not involved in Ca²⁺-activated Cl⁻ currents. The outward Cl⁻ currents at +69 mV were inhibited by NPPB (100 μm), IAA-94 (100 μm), DIDS (0.03–1 mm), 9-AC (300 μm and 1 mm) and DPC (1 mm), whereas the inward currents at −101 mV were not. These results demonstrate the presence of a bicarbonate- and weak acid-permeable Cl⁻ conductance controlled by cytosolic Ca²⁺ and ATP levels in rat submandibular acinar cells. Received: 9 January 1996/Revised: 20 May 1996     

肾素－血管紧张素系统──应激激素反应系统

各种急性与慢性应激时,循环血中及脑、心血管、肾腺等组织中血管紧张素Ⅱ（ＡⅡ）显著增多;人剧烈运动后血浆ＡⅡ与皮质醇一样均剧烈增加,在慢性应激性高血压动物循环及组织中,ＡⅡ含量持续地增高。还发现高浓度ＡⅡ对肾上腺糖皮质激素的分泌有直接的刺激作用,而组织ＡⅡ增多又受肾上腺素能β受体的激发。根据近年来的系列工作,并结合文献,我们认为ＡⅡ是一种重要的应激激素;肾素－血管紧张素系统是一个应激激素反应系统;它与经典的应激激素反应系统,下丘脑－垂体－肾上腺皮质系统及交感－肾上腺髓质系统也存在着密切的关系。     

Immunocytochemical localization of Na+/K+-ATPase and V-H+-ATPase in the salivary glands of the cockroach,Periplaneta americana

The acinous salivary glands of the cockroach (Periplaneta americana) consist of four morphologically different cell types with different functions: the peripheral cells are thought to produce the fluid component of the primary saliva, the central cells secrete the proteinaceous components, the inner acinar duct cells stabilize the acini and secrete a cuticular, intima, whereas the distal duct cells modify the primary saliva via the transport of water and electrolytes. Because there is no direct information available on the distribution of ion transporting enzymes in the salivary glands, we have mapped the distribution of two key transport enzymes, the Na⁺/K⁺-ATPase (sodium pump) and a vacuolar-type H⁺-ATPase, by immunocytochemical techniques. In the peripheral cells, the Na⁺/K⁺-ATPase is localized to the highly infolded apical membrane surface. The distal duct cells show large numbers of sodium pumps localized to the basolateral part of their plasma membrane, whereas their highly folded apical membranes have a vacuolar-type H⁺-ATPase. Our immunocytochemical data are supported by conventional electron microscopy, which shows electrondense 10-nm particles (portasomes) on the cytoplasmic surface of the infoldings of the apical membranes of the distal duct cells. The apically localized Na⁺/K⁺-ATPase in the peripheral cells is probably directly involved in the formation of the Na⁺-rich primary saliva. The latter is modified by the distal duct cells by transport mechanisms energized by the proton motive force of the apically localized V-H⁺-ATPase.     

Epinephrine, norepinephrine, and cortisol concentrations in cannulated seawater-acclimated rainbow trout (Oncorhynchus mykiss) following black-box confinement and epinephrine injection

The present study investigates the effect of cannulation and chronic'black-box' confinement, as well as epinephrine administration (4–0 μg kg⁻¹), on the degree and time-course of alterations in trout ( Oncorhynchus mykiss ) catecholamine and cortisol concentrations. Plasma cortisol concentrations in seawater trout acclimated to 3–6° C reached 104 ng ml⁻¹ 1 day after cannulation/confinement and remained elevated above resting levels (8 ng ml⁻¹) until 6 days post-confinement. Although plasma epinephrine and norepinephrine generally declined over the period of confinement (day 1 approx. 12 nM; day 7 approx. 6 nM), norepinephrine titres were usually higher and more variable. Epinephrine injection caused elevations in plasma epinephrine levels but not in norepinephrine levels; epinephrine titres reaching 107 ± 26 nM (range 65–238 nM) at 2 min post-injection and returning to pre-injection levels by 30 min post-injection. Plasma cortisol increased by 20 ng ml⁻¹ following epinephrine administration. Based on the time-course for post-confinement alterations in plasma cortisol, it appears that up to a week may be required before cannulated fish are completely acclimated to 'black-box' confinement. The findings suggest that meaningful results from experiments utilizing epinephrine injection and 'black-box confinement are contingent upon: (1) knowledge of circulating epinephrine levels shortly after injection (i.e. within 2 min post-injection); and (2) an experimental design that takes into account the elevated cortisol titres that are inherent with cannulation/confinement and epinephrine injection.     

20-OH-ecdysone swells nuclear volume by alkalinization in salivary glands of Drosophila melanogaster

Ecdysteroids play an important role in the larval moulting process of insects. Ecdysone-induced stimulation causes specific puffs in polytene chromosomes of salivary gland cells resulting in nuclear swelling. During this process, changes of intracellular ion composition are thought to act as an early regulatory mechanism of gene activation. By use of video-imaging analysis and electrophysiological techniques, we examined ecdysone-induced nuclear swelling in Drosophila salivary glands in situ and its dependence on pH and calcium. Isolated glands of the third larval stage were superfused with a solution mimicking the haemolymph. Addition of 5×10^–6 mol/l 20-OH-ecdysone led, after a lag period of 50 min, to a sustained Ca²⁺-dependent increase of nuclear volume by 23.0±2.3%. Amiloride, a blocker of plasma membrane Na⁺/H⁺ exchange, prevented 20-OH-ecdysone-induced nuclear swelling. Decreasing pH in the superfusate from 7.15 to 6.8 led to nuclear shrinkage by 16.9±3.9%. Measurments of pH in salivary gland cells with ion-sensitive microelectrodes disclosed an alkalinization of 0.23±0.05 pH units after stimulation with 20-OH-ecdysone. We postulate that 20-OH-ecdysone activates the amilorde-sensitive plasma membrane Na⁺/H⁺ exchanger. This leads to intracellular alkalinization and concomitant decondensation of the nuclear chromatin visible as nuclear swelling. Thus, cell alkalinization could be a potentially important stimulatory mechanism in mediating ecdysteroid-induced activation of the cell nucleus.