Comparison of in vivo and in vitro survival and fecundity rates of the poultry red mite, Dermanyssus gallinae.

To assist in the testing of possible antigens in developing a vaccine against the poultry red mite (Dermanyssus gallinae De Geer), a rapid and reliable in vitro screening method is critical. This short paper describes how D. gallinae survival and fecundity rates in an in vivo feeding device compared to that of mites fed using an in vitro method. Results showed that survival of fed D. gallinae females and mites overall was greater in vitro, although there was no difference between male survival and fecundity between in vivo and in vitro designs. The in vitro feeding device described therefore has the potential to provide reliable results, comparable to those obtained by in vivo testing, to allow for the rapid screening of D. gallinae antigens.

Regulation and induction of CYP3A11, CYP3A13 and CYP3A25 in C57BL/6J mouse liver.

This study reports that dexamethasone (DEX) significantly induces CYP3A11, CYP3A13 and CYP3A25 mRNA expression in male and female 4 days, 3 weeks and 18 weeks old C57BL/6J mice. Furthermore, CYP3A activity, as measured by erythromycin-N-demethylation, is also significantly increased. PXR, RXRalpha and CAR are known to be involved in the induction of CYP3As. Here we report nuclear receptors PXR and RXRalpha but not CAR demonstrate gender- and age-dependent expression. Also, treatment of C57BL/6J mice with DEX induces PXR but not RXRalpha or CAR. In summary, we demonstrate DEX is not only able to up-regulate CYP3A expression and activity, but also the nuclear receptor PXR through which it may exert this effect. Furthermore, the gender- and age-dependent pattern of basal PXR and RXRalpha expression is similar to the 3 CYP3As analysed.

A comparison of the effects of the 5HT1A antagonists MM-77 and WAY-100635 on the mouse isolated vasa deferentia.

1. Experiments were carried out to characterize the possible adrenergic properties of the 5-HT(1A) antagonists WAY 100635 and MM-77 using the mouse isolated vasa deferentia preparation. 2. When vasa deferentia were preincubated for 10 min in the presence of MM-77 (10(-8)-10(-6) m) or WAY100635 (10(-8)-7 x 10(-7) m), a concentration-dependent inhibition of the contractile response to submaximal electrical field stimulation (10 Hz, 50 V, 50 ms) was observed with pIC(50) values of 7.05 +/- 0.01 and 6.85 +/- 0.1 respectively. 3. MM-77 (10(-8)-10(-6) m) antagonized the contractile responses of the vasa deferentia to phenylephrine (PE) (10(-6)-10(-3) m) in a concentration-dependent manner. Schild plots of these data were linear and yielded a mean rhoA(2) value of 6.81 +/- 0.084. The mean slope was 1.42 +/- 0.22. 4. WAY100635 (10(-8)-10(-6) m) antagonized the contractile responses of the vasa deferentia to PE (10(-6)-10(-3) m) in a concentration-dependent manner. Schild plots of these data were linear and yielded a mean rhoA(2) value of 7.05 +/- 0.08. The mean slope was 0.97 +/- 0.1. 5. The results suggest that while WAY100635 acts as a competitive antagonist at alpha(1)-adrenoceptors, MM-77 displays non-competitive antagonist characteristics at this receptor subtype. 6. These results may have important implications for the use of these compounds as 5-HT(1A) receptor antagonists in in vivo studies.

Estimation of intracellular calcium activity in confluent monolayers of primary cultures of quail medullary bone osteoclasts.

The present study was concerned with an investigation of the relative utilities of the fluorescent indicators fura-2 and fluo-3 for spectrofluorimetric estimation of the intracellular calcium concentration ([Ca2+]i) in confluent osteoclast monolayers that had been isolated from medullary bone of quail hens and maintained in tissue culture for 6-8 days. Additionally, we have determined the effects of raised extracellular calcium ([Ca2+]o) and chicken calcitonin (CT) on [Ca2+]i in this preparation. Relative to fura-2, fluo-3 was poorly incorporated into the osteoclasts and had a high apparent equilibrium binding constant (Kd) for Ca2+ binding (809 nM). The osteoclasts were only weakly sensitive to the calcium ionophore, ionomycin. It is concluded that fura-2 is of greater utility than furo-3 in this preparation. In contrast to its lack of effect in freshly isolated cells, elevated [Ca2+]o up to 20 mM stimulated a concentration-dependent increase in [Ca2+]i in cultured osteoclasts, but CT was without effect. These findings further support the idea that quail osteoclasts are able to acquire a Ca2+ sensor or 'receptor' and thus to respond to [Ca2+]o in a similar manner to mammalian osteoclasts when they are removed from the bone microenvironment, but retain a refractoriness to CT under these conditions.

The relationship of intracellular free calcium activity to amylase secretion in substance P- and isoprenaline-stimulated rat parotid acini.

The relationship between intracellular free calcium activity ([Ca2+]i) and stimulated amylase secretion was investigated in rat parotid acini by measuring the effects of substance P methyl ester and isoprenaline on quin2 fluorescence and amylase release. Although both of these drugs evoked concentration-dependent increases in [Ca2+]i and amylase release, the tachykinin had a greater effect on [Ca2+]i while the catecholamine was the more effective secretagogue. Whereas isoprenaline exerted equipotent effects on amylase secretion and [Ca2+]i, the dose-response relationship for stimulation of secretion by substance P was dissociated by three orders of magnitude to the right of that for elevation of [Ca2+]i by this peptide. It is concluded that these data do not support the hypothesis that substance P-stimulated amylase secretion is mediated solely through an increase in [Ca2+]i and that other second-messengers may be involved in mediation of this secretory response.

Mechanism of the inhibitory effect of trifluoperazine on isoprenaline-evoked amylase secretion from isolated rat parotid glands.

We have investigated the effects of the calmodulin antagonist trifluoperazine (TFP) on amylase secretion and adenosine 3':5'-monophosphate (cyclic AMP) metabolism using incubated parotid glands of young rats. Exposing unstimulated glands to 100 microM TFP doubled the basal rate of amylase and lactate dehydrogenase (LDH) release, but had no effect on either the parotid cyclic AMP or ATP contents. Isoprenaline (1 microM) stimulated amylase secretion and increased the tissue cyclic AMP content. 100 microM TFP inhibited these responses by 46% and 33%, respectively. N6,O2-dibutyryl adenosine 3',5'-monophosphate (dibutyryl cyclic AMP) mimicked the effect of isoprenaline on amylase release but 100 microM TFP had no effect on this response. 10 microM TFP inhibited F- -stimulated adenylate cyclase activity in a subcellular fraction isolated from the parotid by 32%. We conclude that TFP may inhibit isoprenaline-evoked amylase secretion from the rat parotid by an effect on either the catalytic or regulatory subunits of adenylate cyclase.

Morphological, biochemical and secretory studies on rat pancreatic ducts maintained in tissue culture.

Interlobular ducts were isolated from the pancrease of copper-deficient rats and maintained in culture on polycarbonate filter rafts. Within 8 h the ends of the ducts had sealed. This was followed by a marked dilatation of the lumen, a flattening of the epithelium against the surrounding connective tissue layer and an over-all swelling of the duct. Apart from a reduction in their height, a fall in the number of intracellular fat droplets and a widening of intercellular spaces, epithelial cells within the cultured ducts retained all the ultrastructural characteristics of those in freshly isolated preparations. The basal concentration of adenosine 3',5'-phosphate (cyclic AMP) in the cultured ducts was 43.3 +/- 6.8 mumol.1-1 duct epithelium (n = 5) and was increased to 188.9 +/- 55.2 mumol.l-1 duct epithelium (n = 5) in the presence of 10(-8) M secretin. The basal rate of fluid secretion, measured using micropuncture techniques, was 0.16 +/- 0.03 nl.h-1.nl-1 duct epithelium (n = 12). This was increased 14-fold by 10(-8) M secretin while the dose of the hormone required for half-maximal secretion was about 2 X 10(-11) mol.l-1. The concentration of chloride ions in secreted fluid and perifusion buffer were similar. Variation in culture time up to 52 h had no effect on fluid secretion, and the response to secretin was dependent on the presence of bicarbonate ions in the perifusion fluid. N6,O2-dibutyryl adenosine 3',5'-phosphate (dibutyryl cyclic AMP) also increased fluid secretion but caerulein had no effect. We suggest that isolated ducts secrete fluid at comparable rates to ducts in situ within the pancrease of copper-replete rats.

Isolation of ducts from the pancreas of copper-deficient rats.

A technique is described, involving tissue dissociation and micro-dissection, for the isolation of interlobular ducts from the pancreas of copper-deficient rats. The average length and outside diameter of the isolated ducts were 589.0 +/- 18.6 and 78.1 +/- 1.6 micron (mean +/- S.E.M., n = 425) respectively. Between twenty and fifty ducts could be obtained from each pancreas. Frequently, the smaller intralobular ducts, which had outside diameters of between 15 and 25 micron, were observed as branches of the interlobular ducts. Light and electron microscopy showed that the isolated ducts were structurally intact, and that the epithelial cells possessed all the typical ultrastructural features of duct cells within the gland of copper-replete rats. The isolated ducts consumed oxygen at a rate of 2.27 +/- 0.55 ml O2/min X 100 g wet weight duct epithelium (n = 6). The concentrations of ATP, ADP and AMP in the ducts were 3.78 +/- 0.81, 0.68 +/- 0.19 and 0.41 +/- 0.13 mmol/l duct epithelium (n = 8) respectively. These data give values for ATP:ADP and ATP:AMP ratios of 5.6:1 and 9.2:1 respectively, and an energy charge of 0.85 +/- 0.01 (n = 8) suggesting that the epithelial cells are healthy and in a stable metabolic state. In the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.67 mM), the basal concentration of cyclic AMP in the isolated ducts was 17.4 +/- 0.7 mumol/l duct epithelium (n = 3). Secretin (0.1 nM-1 microM) caused a dose-related increase in cyclic AMP content up to a maximum of 376.0 +/- 85.3 mumol/l duct epithelium (n = 4). This indicates that the epithelial cells possess secretin receptors, and that these receptors can be functionally linked to adenylate cyclase.

Mechanism of action of extracellular calcium on isoprenaline-evoked amylase secretion from isolated rat parotid glands.

The effects of extracellular Ca2+ deprivation on amylase secretion, 45Ca efflux and cyclic adenosine 3',5'-monophosphate (cyclic AMP) metabolism were investigated in incubated parotid glands of young rats. Reducing the extracellular Ca2+ concentration from 2.5 X 10(-3) M to 10(-6) M had no effect on amylase secretion but did increase the rate of 45Ca efflux from unstimulated glands. Isoprenaline (10(-5) M) increased amylase secretion and the rate of 45Ca efflux in media containing 2.5 X 10(-3) M-Ca2+, and also increased the parotid cyclic AMP content. When glands were pre-incubated for either 10, 20, or 30 min in media containing either 10(-3) or 10(-6) M-Ca2+ there was no effect on the peak rate of amylase secretion stimulated by isoprenaline but the response took longer to develop. The effects of the same experimental manoeuvre on the net change in 45Ca efflux stimulated by isoprenaline were consistent with the idea that the beta-adrenergic agonist mobilizes Ca2+ from a number of intracellular pools. When glands were pre-incubated for 100 min in Ca2+-depleted buffers and then exposed to isoprenaline a triphasic effect on amylase secretion was observed. The secretory response was depressed when the extracellular Ca2+ concentration was lowered from 2.5 X 10(-3) M to 10(-3) M; remained unaffected when the extracellular Ca2+ was further reduced to 10(-5) M; and was virtually abolished when the extracellular Ca2+ concentration was decreased to 10(-6) M. When glands were pre-incubated for 100 min in media containing Ca2+ at concentrations above 10(-5) M and then stimulated with isoprenaline parallel changes in cyclic AMP content and amylase secretion were observed. At lower Ca2+ concentrations there was no further reduction in cyclic AMP content. The lipophilic cyclic AMP analogue N6,O2-dibutyryl adenosine 3',5'-monophosphate (dibutyryl cyclic AMP) mimicked the effects of isoprenaline on amylase secretion and 45Ca efflux. However, the amylase secretory response took longer to develop and the 45Ca efflux response was of shorter duration. These responses were reduced after pre-incubation of glands for 100 min in a medium containing 10(-6) M-Ca2+. Ca2+ at concentrations up to 10(-7) M enhanced parotid adenylate cyclase activity but higher concentrations were inhibitory. The same pattern of Ca2+ sensitivity was seen for basal and also isoprenaline and F--stimulated activities. The results show that isoprenaline can mobilize Ca2+ from a number of intracellular pools which may exchange at different rates with the extracellular medium. This Ca2+ may be required at a minimum of two sites in the amylase secretory pathway.(ABSTRACT TRUNCATED AT 400 WORDS)