Search

Response curves were established for different supplements, offered at intakes ranging from 0 to 20 g/kg liveweight (W).day to young Bos indicus crossbred steers fed low-quality Rhodes grass (Chloris gayana) hay ad libitum in two pen experiments. Supplements included protein meals of varying rumen-degradability (cottonseed meal (CSM) or fishmeal), as well as 'energy sources' comprising grains of high and low ruminal starch degradability (barley and sorghum) and a highly fermentable sugar source (molasses), with all diets adjusted for rumen-degradable nitrogen and mineral content. Unsupplemented steers gained 0.08 and 0.15 kg/day, in Experiments 1 and 2, respectively. Growth of steers increased linearly with intake of 'energy source' supplements in increasing order of molasses, sorghum and barley (all differences P < 0.05). Steer growth rate also increased linearly with fishmeal, albeit over a narrow intake range (0-4.1 g/kg W.day), whereas the response with CSM was asymptotic, showing a steep response at low intake before levelling at ~1.2 kg/day. All supplement types were associated with a linear reduction in hay intake by the steers (energy substitution) where the reduction was greater (P < 0.05) for barley and molasses (not different) than for sorghum (P < 0.05), and for fishmeal compared with CSM (P < 0.05). In concurrent metabolism studies with the same rations, organic matter digestibility of the total ration (561-578 g/kg DM, unsupplemented) was increased linearly by barley and molasses (both P < 0.05) but was unaffected by CSM and sorghum supplements. The efficiency of microbial protein synthesis in steers increased linearly, from 91 g microbial crude protein/kg digestible organic matter (unsupplemented), in both molasses and CSM-supplemented steers, with the trend for a higher response to molasses (P = 0.05), and appeared most closely related to digestible organic matter intake. The response curves from these studies provide the practical framework upon which to formulate rations for cattle grazing low-quality forages. [ABSTRACT FROM AUTHOR]

A pen feeding study was carried out over 70 days to determine the effects of monensin (M) inclusion in two commercial supplements designed to provide different planes of nutrition to recently weaned steers. Thirty Bos indicus crossbred steers (191.4 ± s.d. 7.1 kg) were individually fed a low quality pangola grass hay (57 g crude protein/kg DM; 497 g/kg DM digestibility) ad libitum (Control) with either a urea/molasses-based supplement of Rumevite Maxi-graze 60 Block (B), fed at 100 g/day, or grain-based Rumevite Weaner Pellets (WP), fed at 7.5 g/kg liveweight (W).day, both with and without M, viz. B, B+M, WP and WP+M, respectively. There were no significant interactions between supplement type and M inclusion for any measurement. Growth rates (main effects) averaged 0.17, 0.35 and 0.58 kg/day for the Control, B and WP supplements, respectively, with all means different (P < 0.05), while the response (P < 0.05) to M across supplement type was 0.11 kg/day. Hay DM intake was similar for the Control and B treatments (18.6 and 19.6 g/kg W.day) but was reduced (P < 0.05) with the WP supplement (16.8 g/kg W.day) while corresponding total DM intakes increased from 18.6 to 20.0 to 23.5 g/kg W.day (all differences P < 0.05), respectively. Monensin inclusion in the supplements did not affect supplement, hay or total DM intake. Inclusion of M in supplements for grazing weaners in northern Australia may increase survival rates although the effect of M with cattle at liveweight maintenance or below requires further investigation. Poor dry season nutrition coupled with diseases such as coccidiosis leads to reduced survival of young cattle in northern Australia. A pen feeding experiment carried out to determine the effect of dietary monensin inclusion showed a growth response of about 0.1 kg/day in steers fed supplements promoting either a low or medium plane of nutrition. Monensin has a key role in improving the nutrition and welfare of young grazing cattle. [ABSTRACT FROM AUTHOR]

In this paper we describe the properties of the secretagogue-evoked chloride current from mouse pancreatic acinar cells. Single cells were patch-clamped in the whole-cell configuration with solutions that excluded cation currents and then stimulated with 1 mM carbachol (CCh). This resulted in a current that rose to a peak and then decayed to a plateau level. The current/voltage relationship of the peak current was linear whereas that of the plateau phase was rectified and showed time and voltage dependence. To determine if the CCh evoked current was strictly Ca2+ dependent, we compared the properties of the plateau current with those of currents evoked by directly raising cytosolic [Ca2+]. The properties of the two currents were the same, with both currents showing outward rectification, development at depolarised potentials, similar time constants of activation, permeability sequences and sensitivity to the Cl– channel inhibitor 4,4′-diisothiocyanatodihydrostilbene-2,2′-disulphonic acid (DIDS). We conclude that the agonist-evoked rise in Ca2+ is alone a sufficient signal to activate the CL– current. [ABSTRACT FROM AUTHOR]

Focuses on the diversity of calcium-dependent chloride channels and the mechanisms underlying their calcium-dependent activation. Details of the distribution and characterization of the channels; Structure of the local secretory pole calcium signal; Future research implications of the secretory process.

In this paper we describe the properties of the secretagogue-evoked chloride current from mouse pancreatic acinar cells. Single cells were patch-clamped in the whole-cell configuration with solutions that excluded cation currents and then stimulated with 1 mM carbachol (CCh). This resulted in a current that rose to a peak and then decayed to a plateau level. The current/voltage relationship of the peak current was linear whereas that of the plateau phase was rectified and showed time and voltage dependence. To determine if the CCh evoked current was strictly Ca2+ dependent, we compared the properties of the plateau current with those of currents evoked by directly raising cytosolic [Ca2+] The properties of the two currents were the same, with both currents showing outward rectification, development at depolarised potentials, similar time constants of activation, permeability sequences and sensitivity to the Cl- channel inhibitor 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulphonic acid (DIDS). We conclude that the agonist-evoked rise in Ca2+ is alone a sufficient signal to activate the CL- current.

In secretory epithelial cells, complex patterns of Ca2+ signals regulate physiological processes. How these patterns are generated is still not fully understood. In particular, the basis of global Ca2+ waves is not clear. We have studied regional differences in InsP3-evoked Ca2+ release in single mouse pancreatic acinar cells, using high-speed (approximately 90 frames s-1), high-sensitivity Ca2+ imaging combined with rapid (10 ms) spot photolysis (2 micrometer diameter) of caged InsP3. Within a single region we measured Ca2+ response latency and rate of rise to construct an InsP3 dose-response relationship. Spot InsP3 liberation in the secretory pole region consistently elicited a dose-dependent, rapid release of Ca2+. Spot InsP3 liberation in the basal pole region of approximately 50% of cells elicited a similar dose-response relationship but with a lower apparent InsP3 affinity than in the secretory pole. In the other cells, basal pole InsP3 liberation did not elicit active Ca2+ release, even at the highest stimulus intensities we employed, although these same cells did respond when the stimulus spot was moved to different regions. We conclude that in the basal pole active sites of rapid Ca2+ release have a lower functional affinity for InsP3 than those in the secretory pole and are spread out in discrete sites across the basal pole. These properties explain the propagation of Ca2+ waves across the basal pole that are only observed at higher stimulus levels.

The role of the cytoskeleton in regulating Ca(2+) release has been explored in epithelial cells. Trains of local Ca(2+) spikes were elicited in pancreatic acinar cells by infusion of inositol trisphosphate through a whole cell patch pipette, and the Ca(2+)-dependent Cl(-) current spikes were recorded. The spikes were only transiently inhibited by cytochalasin B, an agent that acts on microfilaments. In contrast, nocodazole (5-100 micrometer), an agent that disrupts the microtubular network, dose-dependently reduced spike frequency and decreased spike amplitude leading to total blockade of the response. Consistent with an effect of microtubular disruption, colchicine also inhibited spiking but neither Me(2)SO nor beta-lumicolchicine, an inactive analogue of colchicine, had any effect. The microtubule-stabilizing agent, taxol, also inhibited spiking. The nocodazole effects were not due to complete loss of function of the Ca(2+) signaling apparatus, because supramaximal carbachol concentrations were still able to mobilize a Ca(2+) response. Finally, as visualized by 2-photon excitation microscopy of ER-Tracker, nocodazole promoted a loss of the endoplasmic reticulum in the secretory pole region. We conclude that microtubules specifically maintain localized Ca(2+) spikes at least in part because of the local positioning of the endoplasmic reticulum.

InsP(3)-evoked elementary Ca(2+) release events have been postulated to play a role in providing the building blocks of larger Ca(2+) signals. In pancreatic acinar cells, low concentrations of acetylcholine or the injection of low concentrations of InsP(3) elicit a train of spatially localized Ca(2+) spikes. In this study we have quantified these responses and compared the Ca(2+) signals to the elementary events shown in Xenopus oocytes. The results demonstrate, at the same concentrations of InsP(3), Ca(2+) signals consisting of one population of small transient Ca(2+) release events and a second distinct population of larger Ca(2+) spikes. The signal mass amplitudes of both types of events are within the range of amplitudes for the elementary events in Xenopus oocytes. However, the bimodal Ca(2+) distribution of Ca(2+) responses we observe is not consistent with the continuum of event sizes seen in Xenopus. We conclude that the two types of InsP(3)-dependent events in acinar cells are both elementary Ca(2+) signals, which are independent of one another. Our data indicate a complexity to the organization of the Ca(2+) release apparatus in acinar cells, which might result from the presence of multiple InsP(3) receptor isoforms, and is likely to be important in the physiology of these cells.

1. The isoflavone genistein may either stimulate or inhibit cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. To investigate how genistein inhibits CFTR, we studied CFTR Cl- channels in excised inside-out membrane patches from cells expressing wild-type human CFTR. 2. Addition of genistein (100 microM) to the intracellular solution caused a small decrease in single-channel current amplitude (i), but a large reduction in open probability (Po). 3. Single-channel analysis of channel block suggested that genistein (100 microM) may inhibit CFTR by two mechanisms: first, it may slow the rate of channel opening and second, it may block open channels. 4. Acidification of the intracellular solution relieved channel block, suggesting that the anionic form of genistein may inhibit CFTR. 5. Genistein inhibition of CFTR Cl- currents was weakly voltage dependent and unaffected by changes in the extracellular Cl- concentration. 6. Channel block was relieved by pyrophosphate (5 mM) and ATP (5 mM), two agents that interact with the nucleotide-binding domains (NBDs) of CFTR to greatly stimulate channel activity. 7. ATP (5 mM) prevented the genistein-induced decrease in Po, but was without effect on the genistein-induced decrease in i. 8. The genistein-induced decrease in i was voltage dependent, whereas the genistein-induced decrease in Po was voltage independent. 9. The data suggest that genistein may inhibit CFTR by two mechanisms. First, it may interact with NBD1 to potently inhibit channel opening. Second, it may bind within the CFTR pore to weakly block Cl- permeation.

1. Cytosolic Ca2+ has been proposed to act as both a positive and a negative feedback signal on the inositol trisphosphate (InsP3) receptor. However, it is unclear how this might affect the Ca2+ response in vivo. 2. Mouse pancreatic acinar cells were whole-cell patch clamped to record the Ca2+-dependent chloride (Cl(Ca)) current spikes and imaged to record the cytosolic Ca2+ spikes elicited by the injection of Ins(2,4,5)P3. Increasing concentrations of Ca2+ buffer (up to 200 microM EGTA or BAPTA) were associated with the appearance of steps in the current activation phase and a prevalence of smaller-amplitude Cl(Ca) spikes. Imaging experiments showed that with increased buffer the secretory pole cytosolic Ca2+ signal became fragmented and spatially discrete Ca2+ release events were observed. 3. At higher buffer concentrations (200-500 microM), increasing concentrations of EGTA increased spike frequency and reduced spike amplitude. In contrast, BAPTA decreased spike frequency and maintained large spike amplitudes. 4. We conclude that, during InsP3-evoked spiking, long-range Ca2+ feedback ( approximately 2-4 microm) shapes the rising phase of the Ca2+ signal by acting to co-ordinate discrete Ca2+ release events and short-range ( approximately 40 nm) Ca2+ feedback acts to inhibit further Ca2+ release.

1. We investigated the effect of protein kinases and phosphatases on murine cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, expressed in Chinese hamster ovary (CHO) cells, using iodide efflux and the excised inside-out configuration of the patch-clamp technique. 2. The protein kinase C (PKC) activator, phorbol dibutyrate, enhanced cAMP-stimulated iodide efflux. However, PKC did not augment the single-channel activity of either human or murine CFTR Cl- channels that had previously been activated by protein kinase A. 3. Fluoride, a non-specific inhibitor of protein phosphatases, stimulated both human and murine CFTR Cl- channels. However, calyculin A, a potent inhibitor of protein phosphatases 1 and 2A, did not enhance cAMP-stimulated iodide efflux. 4. The alkaline phosphatase inhibitor, (-)-bromotetramisole augmented cAMP-stimulated iodide efflux and, by itself, stimulated a larger efflux than that evoked by cAMP agonists. However, (+)-bromotetramisole, the inactive enantiomer, had the same effect. For murine CFTR, neither enantiomer enhanced single-channel activity. In contrast, both enantiomers increased the open probability (Po) of human CFTR, suggesting that bromotetramisole may promote the opening of human CFTR. 5. As murine CFTR had a low Po and was refractory to stimulation by activators of human CFTR, we investigated whether murine CFTR may open to a subconductance state. When single-channel records were filtered at 50 Hz, a very small subconductance state of murine CFTR was observed that had a Po greater than that of human CFTR. The occupancy of this subconductance state may explain the differences in channel regulation observed between human and murine CFTR.

In 20 adult patients (18 male) who presented for thoracotomy, the trachea was intubated using Mallinckrodt disposable double-lumen tubes (18 large and two medium). The endobronchial cuff was inflated by a trained operating department assistant using an air-filled syringe. The volume of air and the initial endobronchial cuff pressure were measured. The minimum cuff pressure required to prevent respiratory gas leakage from the isolated lung was measured also and maintained using the Cardiff Cuff Controller. Mean initial cuff pressure was 69.3 (SEM 6.0) mm Hg, whereas the mean minimum cuff pressure was 29.5 (4.0) mm Hg (P < 0.0001). The results suggest that the method described of inflating the endobronchial cuff may lead to overinflation and subsequent excessive pressure on the endobronchial wall.