Relationship between beta-adrenergic agonists, calpain system activity and beef texture: A systematic review.

The objective was to conduct a systematic review to evaluate the effects of dietary supplementation with beta-adrenergic agonists on calpains and calpastatin activity in bovine muscle and changes in meat tenderness. A survey was conducted in June 2019 on Science Direct, Web of Science, Scopus, PubMed and Capes Periodicals, using four keyword combinations: agonist and calpain and cattle; agonist and calpain and bovine; agonist and calpain and heifers; agonist and calpain and steers. Thirteen studies were selected, 54% concluded that supplementation with beta-adrenergic agonists increases calpastatin activity, 23% observed increase in their gene expression and 23% reported no effect on activity or expression of this enzyme. Nine studies evaluated the influence of beta-adrenergic agonists supplementation on meat texture and all found an increase in shear force values. There is strong evidence that beta-adrenergic agonists may increase calpastatin activity in the muscle, causing damage to meat tenderness.

Phosphoproteomics of Acute Cell Stressors Targeting Exercise Signaling Networks Reveal Drug Interactions Regulating Protein Secretion.

Exercise engages signaling networks to control the release of circulating factors beneficial to health. However, the nature of these networks remains undefined. Using high-throughput phosphoproteomics, we quantify 20,249 phosphorylation sites in skeletal muscle-like myotube cells and monitor their responses to a panel of cell stressors targeting aspects of exercise signaling in vivo. Integrating these in-depth phosphoproteomes with the phosphoproteome of acute aerobic exercise in human skeletal muscle suggests that co-administration of ß-adrenergic and calcium agonists would activate complementary signaling relevant to this exercise context. The phosphoproteome of cells treated with this combination reveals a surprising divergence in signaling from the individual treatments. Remarkably, only the combination treatment promotes multisite phosphorylation of SERBP1, a regulator of Serpine1 mRNA stability, a pro-fibrotic secreted protein. Secretome analysis reveals that the combined treatments decrease secretion of SERPINE1 and other deleterious factors. This study provides a framework for dissecting phosphorylation-based signaling relevant to acute exercise.

Effects of ractopamine hydrochloride and zilpaterol hydrochloride supplementation on longissimus muscle shear force and sensory attributes of calf-fed Holstein steers.

The effect of ractopamine hydrochloride (RH) and zilpaterol hydrochloride (ZH) on slice shear force (SSF) and sensory characteristics of beef from calf-fed Holstein steers was evaluated. All steers were implanted with a progesterone (100 mg) plus estradiol benzoate (10 mg) implant followed by a terminal trenbolone acetate (200 mg) plus estradiol (40 mg) implant. Steers were blocked by weight into pens (n = 32) randomly assigned to 1 of 4 treatments: control, RH fed at 300 mg·steer(-1)·d(-1) (RH 300) or RH fed at 400 mg·steer(-1)·d(-1)(RH 400) for the final 31 d of finishing, or ZH fed at 6.8 g/t for 21 d with a 5-d withdrawal before harvest. Fourteen carcasses were randomly selected from each pen, and two LM samples (1 per side) were excised and aged either 14 or 21 d before SSF testing. For trained panel evaluation, two steaks were collected from each of 60 low Choice strip loins (20 each from control, RH 300, and ZH treatments) and aged either 14 or 21 d. Steers fed RH and ZH produced steaks with SSF values that were 9% to 25% higher than controls. No difference in SSF was detected between the two levels of RH (P > 0.05). Compared to controls, the probability of steaks aged 14 d failing to meet SSF requirements to be certified tender (SSF < 20 kg) was increased 0.15, 0.17, and 0.26 in steers fed RH 300, RH 400, and ZH, respectively. Compared to controls, the probability of steaks aged 21 d having SSF values >20 kg was increased 0.03, 0.08, and 0.16 in steers fed RH 300, RH 400, and ZH, respectively. Steaks from Select carcasses of steers fed ZH aged 21 d postmortem had double the probability (0.39 vs. 0.17) of having SSF values >20 kg compared to steaks from steers fed either level of RH (P < 0.05). This difference tended to be identical in steaks from Select carcasses 14 d postmortem (0.50 vs. 0.33; P = 0.11); however, no difference was found in low Choice samples at 14 or 21 d postmortem. Trained panelists rated steaks aged 14 d from steers fed ZH lower for overall tenderness and flavor compared to controls (P < 0.05); however, no difference was found between controls and those fed RH 300. Steaks from steers fed ZH aged 21 d were rated lower for overall tenderness and juiciness compared to controls and those from steers fed RH 300 (P < 0.05). This study suggests RH and ZH negatively impact sensory attributes of beef from calf-fed Holstein steers.

Effects of ractopamine hydrochloride and zilpaterol hydrochloride supplementation on carcass cutability of calf-fed Holstein steers.

Effects of ractopamine hydrochloride (RH) and zilpaterol hydrochloride (ZH) on saleable yield of carcass sides from calf-fed Holstein steers were evaluated using steers implanted with a progesterone (100 mg) plus estradiol benzoate (10 mg) implant followed by a terminal trenbolone acetate (200 mg) plus estradiol (40 mg) implant. Steers were blocked by weight into pens (n = 32) randomly assigned to one of four treatments: control, RH fed at 300 mg•steer(-1)/d(-1) (RH 300) or RH fed at 400 mg•steer(-1)/d(-1) (RH 400) the final 31 d of finishing, and ZH fed at 60 to 90 mg•steer(-1)/d(-1) (7.56 g/ton on a 100% DM basis) for 21 d with a 5 d withdrawal before harvest. Eight to nine carcass sides were randomly selected from each pen; carcass sides with excessive hide pulls, fat pulls or bruises were avoided. Cutout data were collected within a commercial facility using plant personnel to fabricate sides at a rate of one every 3 to 4 min into items typically merchandised by the facility. All lean, fat and bone were weighed and summed back to total chilled side weight with a sensitivity of ± 2% to be included in the data set. Compared to controls, ß-agonists increased saleable yield of whole-muscle cuts by 0.61%, 0.86% and 1.95% for RH 300, RH 400 and ZH, respectively (P < 0.05). Percent fat was less in carcasses from the ZH treatment compared to controls (P < 0.05); however, this difference was not observed between RH treatments and controls (P > 0.05). Percent bone was less in the ZH treatment due to increased muscle (P < 0.05). The percent of chilled side weight comprised of trimmings was unchanged between treatments, but on a 100% lean basis, RH 400 and ZH increased trim yields (P < 0.05). Analysis of saleable yield by primal showed a fundamental shift in growth and development. Beta-agonists caused a shift in proportion of saleable yield within individual primals, with a greater portion produced from the hindquarter relative to the forequarter, specifically in those muscles of the round (P < 0.05). Beta-agonists increased saleable yield, but these effects were not constant between all major primals. The cutout value gained by packers as a result of ß-agonist use may be influenced more by reduced fatness and increased absolute weight if musculature is primarily increased in the lower priced cuts of the carcass.

Effects of dietary L-carnitine and ractopamine HCl on the metabolic response to handling in finishing pigs.

Two experiments (384 pigs; C22 × L326; PIC) were conducted to determine the interactive effect of dietary L-carnitine and ractopamine HCl (RAC) on the metabolic response of pigs to handling. Experiments were arranged as split-split plots with handling as the main plot and diets as subplots (4 pens per treatment). Dietary L-carnitine (0 or 50 mg/kg) was fed from 36.0 kg to the end of the experiments (118 kg), and RAC (0 or 20 mg/kg) was fed the last 4 wk of each experiment. At the end of each experiment, 4 pigs per pen were assigned to 1 of 2 handling treatments. Gently handled pigs were moved at a moderate walking pace 3 times through a 50-m course and up and down a 15° loading ramp. Aggressively handled pigs were moved as fast as possible 3 times through the same course, but up and down a 30° ramp, and shocked 3 times with an electrical prod. Blood was collected immediately before and after handling in Exp. 1 and immediately after and 1 h after handling in Exp. 2. Feeding RAC increased (P < 0.01) ADG and G:F, but there was no effect (P > 0.10) of L-carnitine on growth performance. In Exp. 1 and 2, aggressive handling increased (P < 0.01) blood lactate dehydrogenase (LDH), lactate, cortisol, and rectal temperature and decreased blood pH. In Exp. 1, there was a RAC × handling interaction (P < 0.06) for the difference in pre- and posthandling blood pH and rectal temperature. Aggressively handled pigs fed RAC had decreased blood pH and increased rectal temperature compared with gently handled pigs, demonstrating the validity of the handling model. Pigs fed RAC had increased (P < 0.01) LDH compared with pigs not fed RAC. Pigs fed L-carnitine had increased (P < 0.03) lactate compared with pigs not fed L-carnitine. In Exp. 2, pigs fed RAC had lower (P < 0.02) blood pH immediately after handling, but pH returned to control levels by 1 h posthandling. Lactate, LDH, cortisol, and rectal temperature changes from immediately posthandling to 1 h posthandling were not different (P > 0.10) between pigs fed L-carnitine and those fed RAC, indicating that L-carnitine did not decrease recovery time of pigs subjected to aggressive handling. These results suggest that pigs fed 20 mg/kg of RAC are more susceptible to stress when handled aggressively compared with pigs not fed RAC. Dietary L-carnitine fed in combination with RAC did not alleviate the effects of stress. This research emphasizes the importance of using proper animal handling techniques when marketing finishing pigs fed RAC.

Supplemental vitamin D3 and zilpaterol hydrochloride. I. Effect on performance, carcass traits, tenderness, and vitamin D metabolites of feedlot steers.

Angus × Simmental steers (n = 210; initial BW 314 ± 11 kg) were separated into heavy and light BW blocks and allotted evenly by BW to 6 treatments (3 heavy and 2 light pens per treatment) to determine the effect of supplemental vitamin D3: 0 IU (no D), 250,000 IU for 165 d (long-term D), or 5 × 10(6) IU for 10 d (short-term D) on performance, carcass traits, vitamin D metabolites, and meat tenderness in steers fed either 0 (NZ) or 8.38 mg/kg zilpaterol hydrochloride (ZH) daily for 21 d. Placebo or ZH was added to the diet 24 d, and short-term D was added 13 d before slaughter. Vitamin D3, ZH, and placebo were all removed from the diet 3 d before slaughter. Steers fed ZH tended to have improved overall G:F compared with steers not fed ZH (P < 0.09). Overall performance was not affected by long-term D, with or without ZH (P = 0.11) compared with no D, with or without ZH. Short-term D decreased final BW, ADG, and G:F (P = 0.04) compared with no D, when ZH was not fed. Zilpaterol hydrochloride increased HCW, dressing percentage, and LM area (P < 0.01); and decreased fat thickness, yield grade, and marbling (P < 0.03). Carcass traits were not impacted by long-term D without ZH (P > 0.13), but long-term D with ZH decreased percentage KPH (P < 0.02). Compared with no D, short-term D tended to decrease HCW (P < 0.07), decreased fat thickness (P < 0.01), and tended to increase dressing percentage (P < 0.10) when ZH was not fed, yet did not impact carcass traits when ZH was fed (P < 0.13). Feeding ZH tended to decrease (P < 0.09) LM 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The long-term D treatment increased LM vitamin D3 and 25-hydroxyvitamin D3 (25OHD3) 18- and 5-fold, respectively, when ZH was not fed (P < 0.04) and increased LM 25OHD3 by 4-fold when ZH was fed (P < 0.01). Short-term D increased LM vitamin D3 and 25OHD3 by 52- and 9-fold, respectively, when ZH was not fed (P < 0.01), and by 24- and 9-fold, respectively, when ZH was fed (P < 0.01). Also, short-term D increased LM 1,25(OH)2D3 by 2-fold (P < 0.04) when ZH was fed. Warner-Bratzler shear force (WBSF) was greater for ZH steaks than non-ZH steaks at 7, 14, and 21 d postmortem aging (P < 0.01). Vitamin D did not reduce WBSF (P = 0.18). When ZH was fed, long-term D tended to increase WBSF in steaks aged 21 d (P = 0.06). In conclusion, ZH improved carcass leanness and decreased tenderness, and vitamin D feeding increased vitamin D3 metabolites in LM, but did not improve tenderness in steers fed ZH.

Supplemental vitamin D3 and zilpaterol hydrochloride. II. Effect on calcium concentration, muscle fiber type, and calpain gene expression of feedlot steers.

Two hundred and ten Angus × Simmental steers (initial BW 314 ± 11 kg) were separated into heavy and light BW blocks and allotted evenly by BW to 6 treatments (3 heavy and 2 light pens per treatment) to determine the effect of supplemental vitamin D3: 0 IU (no D), 250,000 IU for 165 d (long-term D), or 5 × 10(6) IU for 10 d (short-term D) on plasma and muscle calcium concentrations and gene expression in steers fed either 0 (NZ) or 8.38 mg/kg (ZH) zilpaterol hydrochloride (ZH) daily for 21 d. Placebo or ZH was added to the diet 24 d, and short-term D was added 13 d before slaughter. Treatments were removed from all diets 3 d before slaughter. Plasma total calcium (Ca(2+)) was determined at study initiation, start of ZH and short-term D feedings, and at vitamin D3 and ZH withdrawal. Both plasma total and ionic Ca(2+) were determined when animals were sent to harvest. Longissimus muscle total and ionic Ca(2+) were determined in meat aged 7 and 4 d postmortem, respectively. When ZH was fed, long-term D decreased plasma total Ca(2+) at slaughter (P < 0.04). Short-term D increased (P < 0.01) plasma total and ionic Ca(2+) at slaughter regardless of ZH inclusion in the diet. Long- and short-term D, with or without ZH, did not affect (P > 0.28) LM total Ca(2+); however, both long- and short-term D increased LM ionic Ca(2+) when ZH was not fed (P < 0.01). Long-term D reduced LM ionic Ca(2+) when ZH was fed (P < 0.02). Neither long- nor short-term D affected PPARα or δ gene expression (P = 0.19) whether or not ZH was fed. Expression of MYH1 and 2A (P < 0.05) but not 2X (P = 0.21) was decreased in steers fed ZH. Long-term D had no effect on MYH2A expression (P = 0.21). Short-term D increased MYH2A expression when ZH was not fed (P < 0.03). Calpain mRNA tended to be lower in steers fed ZH (P = 0.09), but was not affected by long- or short-term D regardless of whether or not ZH was fed (P = 0.39). Expression of calpastatin did not differ with vitamin D supplementation (P = 0.35). In conclusion, ZH decreased oxidative myosin expression, and when combined with long-term D, ZH decreased LM ionic Ca(2+). Moreover, vitamin D3 supplementation did not increase calpain mRNA. These results help explain why vitamin D3 does not improve tenderness in steers fed ZH.

Impact of health management, health treatments, and zilpaterol hydrochloride supplementation on carcass quality, color, and palatability traits in heifers.

Two hundred sixty-eight strip loins were collected from heifers fed at Oklahoma State University in Stillwater, OK. In Exp. 1, heifers (n = 127) were assigned to 1 of 3 health management treatment groups: antimicrobial administrations were given based on standard feedlot protocol (SFP) or ruminal temperature (RT) or given a metaphylactic treatment of tulathromycin (MT) followed by visual assessment (VA). In Exp. 2, heifers (n = 155) were assigned to the same treatment groups as above and were supplemented zilpaterol hydrochloride (ZH) or control (CON). Three steaks were collected from each strip loin, 1 each for retail display, sensory evaluation, and Warner-Bratzler shear force (WBSF). Color was evaluated from the retail display steak using a trained color panel and objectively using a HunterLab Miniscan XE. An Instron Universal Testing Machine with a Warner-Bratzler head was used for evaluation of instrumental tenderness, and a trained sensory panel was used to assess palatability traits. Heifers treated by VA had the least number of antimicrobial administrations and lowest yield grade and also had the lightest HCW (P < 0.05) compared with the heifers treated by the other health management protocols. There were no subjective color attribute differences or sensory panel differences (P > 0.05) across all health management systems or antimicrobial administrations. There were no differences in carcass and performance traits for any antimicrobial administrations treatment groups (P > 0.05). Heifers who had 0 or 1 antimicrobial administrations had lower (P < 0.05) a* (redness/greenness: positive values = red and negative values = green), and b* (yellowness/blueness: positive values = yellow and negative values = blue) values compared with those who had 2 antimicrobial administrations. In Exp. 2, heifers treated by VA had the least number (P < 0.05) of antimicrobial administrations when compared with MT and RT. Health management group did not have any other effects on carcass, sensory, or color attributes. Zilpaterol hydrochloride supplementation caused a decrease (P < 0.05) in internal fat and yield grade, but no interactions were observed between the number of antimicrobial administrations and ZH supplementation. With the supplementation of ZH, WBSF significantly increased (P < 0.05). At the end of retail display, the control group had a greater (P < 0.05) surface discoloration when compared with the ZH group. Treatment and detection of bovine respiratory diseases (BRD) is critical to the industry economically and results from this study show that different methods can be used to detect BRD without dramatically impacting carcass, sensory, and retail case life characteristics.

Interactive effects of dietary ractopamine HCl and L-carnitine on finishing pigs: I. Growth performance.

A total of 2,152 pigs (C22 × 336 PIC) were used in 4 experiments to determine the interactive effects of dietary l-carnitine and ractopamine HCl (RAC) on finishing pig growth performance. All trials were arranged as factorial arrangements with main effects of l-carnitine (0, 25, or 50 mg/kg in Exp. 1 and 2 and 0 or 50 mg/kg in Exp. 3 and 4) and RAC (0, 5, or 10 mg/kg in Exp. 1 and 0 or 10 mg/kg in Exp. 2, 3, and 4). Dietary carnitine was fed from 38 to 109 kg (Exp. 1 and 3) or for the last 4 or 3 wk before slaughter (118 kg; Exp. 2 and 4, respectively). Ractopamine HCl was fed for 4 wk (Exp. 1, 2, and 3) or 3 wk (Exp. 4) before slaughter. Experiments 1 and 2 were conducted in university research facilities, and Exp. 3 and 4 were conducted in a commercial research facility. All diets were formulated to contain 1.00% total Lys during the last phase of each experiment. In all experiments, pigs fed RAC had increased (P < 0.05) ADG and G:F compared with pigs fed no RAC. Feeding l-carnitine before the RAC feeding period did not affect pig growth performance. In Exp. 1 and 2, l-carnitine did not affect ADG during the last 4 wk; however, in Exp. 2, G:F tended (quadratic; P = 0.07) to improve with increasing l-carnitine. In Exp. 3, l-carnitine × RAC interactions were observed (P < 0.04) for ADG and G:F. Both added l-carnitine and RAC improved performance, but the response was not additive. In Exp. 4, pigs fed l-carnitine had increased (P < 0.04) ADG (0.88 vs. 0.84 kg) and G:F (0.36 vs. 0.35) compared with pigs fed no l-carnitine, and the response was additive to that of RAC. Analysis of treatments common to all experiments showed that pigs fed RAC had increased (P < 0.01) ADG (1.03 vs. 0.93 kg) and G:F (0.40 vs. 0.35) compared with pigs fed no RAC. Pigs fed l-carnitine tended to have increased (P = 0.07) ADG (1.00 vs. 0.96 kg) and improved (P < 0.01) G:F (0.38 vs. 0.37) compared with pigs not fed l-carnitine. These results confirm that RAC improves growth performance of finishing pigs. Added l-carnitine improved growth performance of finishing pigs, and the greatest response was observed in Exp. 3 and 4, which were conducted in commercial research environments. These experiments imply that adding l-carnitine to a finishing diet does not enhance the growth effects of RAC and that effects of RAC and l-carnitine on ADG and G:F are independent.

Interactive effects of dietary ractopamine HCl and L-carnitine on finishing pigs: II. Carcass characteristics and meat quality.

Three experiments using 1,356 pigs (C22 × 336 PIC) were conducted to determine the interactive effects of dietary L-carnitine and ractopamine hydrochloride (RAC) on carcass characteristics and meat quality of finishing pigs. Experiments were arranged as factorials with main effects of L-carnitine and RAC; L-carnitine levels were 0, 25, or 50 mg/kg in Exp. 1 and 2 and 0 or 50 mg/kg in Exp. 3, and RAC levels of 0, 5, or 10 mg/kg in Exp. 1 and 0 or 10 mg/kg in Exp. 2 and 3. Dietary L-carnitine was fed from 38 kg to slaughter (109 and 118 kg in Exp. 1 and 3, respectively) or for 4 wk before slaughter (109 kg in Exp. 2). Ractopamine HCl was fed for 4 wk in all experiments. Exp. 1 and 2 were conducted at university research facilities (2 pigs per pen), and Exp. 3 was conducted in a commercial research barn (23 pigs per pen). In Exp. 1, an L-carnitine × RAC interaction (P < 0.02) was observed for LM visual color, L*, and a*/b*. In pigs fed RAC, increasing L-carnitine decreased L* and increased visual color scores and a*/b* compared with pigs not fed RAC. Ultimate pH tended to increase (linear, P < 0.07) with increasing L-carnitine. Drip loss decreased (linear, P < 0.04) in pigs fed increasing L-carnitine. In Exp. 2, firmness scores decreased in pigs fed increasing L-carnitine when not fed RAC, but firmness scores increased and drip losses decreased with increasing L-carnitine when RAC was added to the diet (L-carnitine × RAC interaction, P < 0.04). Percentage lean was greater (P < 0.01) for pigs fed RAC in Exp. 2. In Exp. 3, fat thickness decreased and lean percentage increased in pigs fed L-carnitine or RAC, but the responses were not additive (L-carnitine × RAC interaction, P < 0.03). Furthermore, pigs fed L-carnitine tended (P < 0.06) to have decreased LM drip loss percentage whereas pigs fed RAC had decreased (P < 0.05) 10th rib and average backfat and decreased drip loss than pigs fed diets without RAC. These results suggest that dietary RAC increased carcass leanness and supplemental L-carnitine reduced LM drip loss when fed in combination with RAC.

Effects of postmortem calcium chloride injection on meat palatability traits of strip loin steaks from cattle supplemented with or without zilpaterol hydrochloride.

An experiment was conducted to determine the effects of zilpaterol hydrochloride mM supplementation (ZH; 8.3 mg/kg on a DM basis for 20 d) and calcium chloride injection [CaCl(2), 200 at 5% (wt/wt) at 72 h postmortem] on palatability traits of beef (Bos taurus) strip loin steaks. Select (USDA) strip loins were obtained from control (no ZH = 19) and ZH-supplemented carcasses (n = 20). Right and left sides were selected alternatively to serve as a control (no INJ) or CaCl(2)-injected (INJ) and stored at 4°C. Before injecting the subprimals (72 h postmortem), 2 steaks were cut for proximate, sarcomere length, and myofibrillar fragmentation index (MFI) analyses. At 7 d postmortem each strip loin was portioned into steaks, vacuum packaged, and aged for the appropriate period for Warner-Bratzler shear force (WBSF; 7, 14, 21, and 28 d postmortem), trained sensory analysis (14 and 21 d postmortem), purge loss (7 d), and MFI (3, 7, 14, 21, and 28 d postmortem). Results indicated steaks from both ZH supplementation and INJ had reduced WBSF values as days of postmortem aging increased. The WBSF values of ZH steaks were greater (P < 0.05) than no ZH steaks at each postmortem aging period. The INJ steaks had lower WBSF values (P < 0.05) than non-injected steaks. A greater percentage (91 vs. 71%) of steaks had WBSF values < 4.6 kg from steers with no ZH supplementation at 7 d postmortem, but the percentage did not differ (P > 0.05) due to ZH at 14, 21, or 28 d or due to INJ at any aging period. Trained panelists rated tenderness less in ZH steaks than steaks with no ZH at 14 d and 21 d. However, INJ improved (P < 0.05) the tenderness ratings and flavor intensity of the trained panelists, compared with their non-injected cohorts at 21 d. Zilpaterol hydrochloride supplementation reduced (P < 0.05) MFI values, but INJ resulted in greater (P < 0.05) MFI values compared with no INJ. Subprimals from ZH and INJ showed greater purge loss (P < 0.05). Although no interactions were found with ZH and CaCl(2), injecting USDA Select strip loins from ZH-fed cattle can help reduce the normal WBSF variation as it does in steaks from non-ZH-fed cattle.

Mutagenesis of important amino acid reveals unconventional homologous internalization of beta(1)-adrenergic receptor.

AIMS: The study was designed to examine the internalization of Asp104Lys mutant of beta(1)-adrenergic receptor (beta(1)-AR) and compared to other mutant (Asp104Ala) and wild type receptors. Moreover, this study needs to perform the role of GRK2 (betaARK1) and beta-arrestin1 on this internalization of Asp104Lys mutant of beta(1)-AR. MAIN METHODS: Binding affinity, functional potency of agonist and agonist-induced internalization were determined for wild type and both mutants of beta(1)-ARs stably expressed in HEK 293 cells as assessed by [(3)H] CGP12177 radioligand. We have performed GRK2 and beta-arrestin1 expression levels by western blot analysis and also performed internalization of this mutant receptor after over expression and deletion of beta-arrestin1 gene. KEY FINDINGS: In the present study, the binding affinity of (-)-isoproterenol for both mutants were significantly decreased compared to wild type. Though the mutant Asp104Ala showed agonist-induced receptor activation, interestingly this mutant was not internalized. However, the mutant Asp104Lys, which showed uncoupling with G protein, was internalized 31.77+/-3.13% from cell surface. Asp104Lys mutant produced the same level of GRK2 expression in (-)-isoproterenol induced stimulation of wild type receptor and addition of (-)-isoproterenol further increased GRK2 expression in mutant receptors. In addition, overexpression of beta-arrestin1 in mutant Asp104Lys promoted (39.75+/-2.19%) and knockdown of beta-arrestin1 by siRNA decreased (3.55+/-1.75%) internalization compared to Asp104Lys mutant of beta(1)-ARs. SIGNIFICANCE: The present studies suggest that Asp104Lys mutant beta(1)-ARs triggers unconventional homologous internalization induced by G protein independent signals, where GRK2 and beta-arrestin1 play an important role for beta(1)-AR internalization.

Inhibitory effects of various beverages on human recombinant sulfotransferase isoforms SULT1A1 and SULT1A3.

Sulfotransferase (SULT) 1A1 and SULT1A3 play important roles in the presystemic inactivation of beta(2) agonists in the liver and intestine, respectively. The study aimed to investigate the inhibitory effects of grapefruit juice, orange juice, green tea, black tea and oolong tea and their constituents on the activities of SULT1A1 and SULT1A3. The activities of both SULT1A1 and SULT1A3 were significantly inhibited by all the beverages investigated at a concentration of 10%. The beverage constituents were tested in concentration ranges considered to be physiologically relevant. The grapefruit constituent, quercetin, completely inhibited SULT1A1, while quercetin and naringin both partially inhibited SULT1A3. The orange constituents, tangeretin and nobiletin, also completely inhibited SULT1A1. The tea constituents, (-)-epicatechin gallate and (-)-epigallocatechin gallate, both almost completely inhibited SULT1A1 and SULT1A3. Moreover, the theaflavin and thearubigin fractions of black tea both completely inhibited SULT1A1 and strongly inhibited SULT1A3. The inhibitory action of green tea on SULT1A3 was competitive, while that of black tea and oolong tea was mixed competitive/non-competitive. Mechanism-based inhibition was not observed with any beverage. In conclusion, various beverages, especially teas, inhibit the function of SULT1A3, and therefore may have the potential to increase the bioavailability of orally administered substrates of SULT1A3, such as beta(2) agonists.

A comparison of two exercise training programs on cardiac responsiveness to beta-stimulation in obesity.

We demonstrated previously that exercise training did not restore normal cardiac beta-adrenergic responsiveness in obese rabbits. This study tested the hypothesis that an increased training volume was required to attenuate obesity-related reductions in isolated heart responsiveness to isoproterenol. Female New Zealand White rabbits were divided into lean control, lean exercise-trained, obese control, and obese exercise-trained groups. For the exercise-trained groups, total treadmill work over 12 weeks was increased 27% when compared with lean and obese animals trained with lower total training volume. After 12 weeks, Langendorff isolated hearts were used to study developed pressure, +dP/dt(max), and -dP/dt(max) responses to isoproterenol (10(-9) - 3 x 10(-7) M). Concentration-response data were fit to a sigmoidal function using a four-parameter logistic equation. Controls were compared with animals trained under the low- and high-training volume programs using one-way analysis of variance and Tukey's post-hoc test; separate analyses were conducted for lean and obese rabbits. In both lean and obese groups trained under the high-training volume program, EC50 values for +dP/day(tmax) and -dP/dt(max) were higher compared with same-weight controls and animals trained under the low-training volume program, indicating that contractility and relaxation responsiveness to isoproterenol was reduced by the higher training volume. Therefore, these data indicate that increased training volume failed to attenuate obesity-related decrements in isolated heart responsiveness to beta-adrenergic stimulation and caused reduced sensitivity to isoproterenol in both lean and obese animals.

[Study on the interactions between Ligusticum chuanxiong extract and cardiac muscle membrane receptors by CMSP chromatography].

OBJECTIVE: To study the interactions between Ligusticum chuanxiong Hort extract and cardiac muscle membrane receptors. METHOD: The cell membrane of rabbit cardiac muscle was fixed on silicon to make cell membrane stationary phase (CMSP), and then the interactions were studied by comparing the retention characteristics of the extracts from different solvents with those of the antagonists or activators corresponding to known receptors in cardiac muscle membrane, and by competition effect on the retention characteristics of extracts when adding the antagonists or activators into the mobile phase. RESULT: Water extract and ethanol extract both had retentions on CMSP; the retention characteristics of water extract could be affected when water extract was in competition with the antagonists for alpha receptor, and could not be affected when with the activator beta1 receptor. CONCLUSION: It is possible that some components in water extract may combine with alpha receptor and no component with beta1 receptor, and that some components in ethanol extract may combine with cardiac muscle cell membrane. The process between active components and receptors in vivo can be imitated through the interactions between drugs and CMSP. The method provides references for the resolution of two applications: to screen the active components from Chinese medicine, and to figure out the type of receptors involved.

Beta-2 mimetics and magnesium: true or false friends?

Physiological beta stimulation may be involved in the regulation of magnesium status namely by homeostatic increase of magnesemia during magnesium deficiency. But conversely excessive beta stimulation namely by use of pharmacological high doses of beta mimetics may induce a decrease of magnesemia. Two different types of magnesium therapy ought to be distinguished. Nutritional magnesium therapy which may physiologically palliate a magnesium deficiency due to an insufficient magnesium intake. It is devoid of any toxicity. Pharmacological magnesium therapy, whatever the magnesium status, causes a iatrogenic magnesium load. It may induce magnesium toxicity. Tocolysis is the one common obstetrical indication for beta mimetics and magnesium. Beta-2 mimetics are the reference tocolytic drugs in most countries. But high doses of beta-2 mimetics for suppression of premature labor are associated to a high incidence of maternal, fetal and neonatal side effects. Tocolysis must then be discontinued or limited to shorter treatments with the lowest possible doses. Nutritional magnesium therapy which palliates gestational magnesium deficiency is efficient and atoxic. Conversely, high doses of intravenous MgSO4 for tocolysis are less efficient and unsafe. Because of its maternal and above all pediatric side effects, this maternal pharmacological magnesium therapy should be abandoned for tocolysis. Investigation of the therapeutic ratio of various magnesium salts before their clinical use could help to determine if other anions different from sulfate could decrease the toxicity. Beta-2 agonists are first line asthma therapy, but their safety is debated. Asthma and Chronic Obstructive Pulmonary Disease (COPD) per se may induce magnesium depletion related to a dysregulation of the control mechanisms of magnesium status. It requires a correction of its causal regulation, but nutritional magnesium supplementation is ineffective. When chronic primary magnesium deficiency coexists with obstructive bronchial disorders, it constitutes a decompensatory factor. Atoxic nutritional magnesium therapy may palliate this coexistent magnesium deficiency. Pharmacological magnesium treatment for obstructive pulmonary diseases is not very efficient with low safety. Combination of palliating nutritional magnesium therapy and of beta-2 mimetics for tocolysis or pulmonary obstructive indications may be beneficial and remain atoxic. Conversely combination of intravenous tocolytic high doses of magnesium and of beta-2 mimetics is contra-indicated because of its dubious efficiency and its possible toxicity. The possible role of SO4- as regards toxicity must be discussed. Contra-indications of lower intravenous or inhaled Mg doses for pulmonary bronchial obstruction are less imperative than for tocolysis. The selection of a particular magnesium salt among others should take into account reliable plasmacological and toxicological data. It seems necessary to determine the therapeutic ratio (LD50/ED50) of the various available magnesium salts before pharmacological use.

The pharmacokinetics of a thiazole benzenesulfonamide beta 3-adrenergic receptor agonist and its analogs in rats, dogs, and monkeys: improving oral bioavailability.

The pharmacokinetics and oral bioavailability of (R)-N-[4-[2-[[2-hydroxy-2-(pyridin-3-yl)ethyl]amino]ethyl]phenyl]-4-[4-[4-(trifluoromethylphenyl]thiazol-2-yl]benzenesulfonamide (1), a 3-pyridyl thiazole benzenesulfonamide beta3-adrenergic receptor agonist, were investigated in rats, dogs, and monkeys. Systemic clearance was higher in rats (approximately 30 ml/min/kg) than in dogs and monkeys (both approximately 10 ml/min/kg), and oral bioavailability was 17, 27, and 4%, respectively. Since systemic clearance was 25 to 40% of hepatic blood flow in these species, hepatic extraction was expected to be low, and it was likely that oral bioavailability was limited either by absorption or a large first-pass effect in the gut. The absorption and excretion of 3H-labeled 1 were investigated in rats, and only 28% of the administered radioactivity was orally absorbed. Subsequently, the hepatic extraction of 1 was evaluated in rats (30%) and monkeys (47%). The low oral bioavailability in rats could be explained completely by poor oral absorption and hepatic first-pass metabolism; in monkeys, oral absorption was either less than in rats or first-pass extraction in the gut was greater. In an attempt to increase oral exposure, the pharmacokinetics and oral bioavailability of two potential prodrugs of 1, an N-ethyl [(R)-N-[4-[2-[ethyl[2-hydroxy-2-(3-pyridinyl)ethyl]amino]ethyl]phenyl]-4-[4-[4-(trifluoromethyl)phenyl]thiazol-2-yl]benzenesulfonamide; 2] and a morpholine derivative [(R)-N-[4-[2-[2-(3-pyridinyl)morpholin-4-yl]ethyl]phenyl]-4-[4-[4-(trifluoromethyl)- phenyl]thiazol-2-yl]benzenesulfonamide; 3], were evaluated in monkeys. Conversion to 1 was low (<3%) with both derivatives, and neither entity was an effective prodrug, but the oral bioavailability of 3 (56%) compared with 1 (4%) was significantly improved. The hypothesis that the increased oral bioavailability of 3 was due to a reduction in hydrogen bonding sites in the molecule led to the design of (R)-N-[4-[2-[[2-hydroxy-2-(pyridin-2-yl)ethyl]amino]ethyl]phenyl]-4-[4-(4-trifluoromethylphenyl)thiazol-2-yl]benzenesulfonamide (4), a 2-pyridyl beta3-adrenergic receptor agonist with improved oral bioavailability in rats and monkeys.

Derivatization procedures for the detection of beta(2)-agonists by gas chromatographic/mass spectrometric analysis.

An evaluation of derivatization procedures for the detection of beta(2)-agonists is presented. The study was performed with the beta(2)-agonists bambuterol, clenbuterol, fenoterol, formoterol, salbutamol, salmeterol and terbutaline. Different derivatizating agents were employed, aiming to obtain derivatives with high selectivity to be used in the gas chromatographic/mass spectrometric analysis of beta(2)-agonists in biological samples. Trimethylsilylation was compared with different agents and the role of some catalysts was evaluated. Acylation, combined trimethylsilylation and acylation, and the formation of cyclic methylboronates were also studied. Sterical hindrance caused by different substituents at the nitrogen atom of the beta-ethanolamine lateral chain of beta(2)-agonist molecules is mainly responsible for differences in the abundances of the derivatives obtained. The use of catalysts produces an increase in the derivatization yield, especially for compounds with low steric hindrance (substituents with primary and secondary carbon atoms). The formation of trimethylsilyl (TMS) ethers is not influenced by structural molecular differences when only hydroxy groups are involved in derivatization. Combined trimethylsilylation and acylation showed that compounds with a secondary carbon atom linked to the nitrogen atom form mainly N-TFA-O-TMS derivatives, with a small amount of N-TMS-O-TMS derivatives. Compounds with tert-butyl substituents at the amino group (bambuterol, salbutamol and terbutaline) formed O-TMS derivatives as the main products, although a limited amount of trifluoroacylation at the nitrogen atom also occurred. Cyclic methylboronates were formed with bambuterol, clenbuterol, formoterol, salbutamol and salmeterol. Owing to hydroxy substituents in unsuitable positions for ring formation, this procedure was not effective for fenoterol and terbutaline. Mass spectra of different derivatives and tentative fragmentation profiles are also shown. For screening purpose (e.g. sports drug testing), derivatization with MSTFA or BSTFA alone is recommended as a comprehensive derivatization technique for beta(2)-agonists owing to minimal by-product formation; formation of cyclic methylboronates can be useful for confirmation purposes. Detection limits were obtained for the TMS and cyclic methylboronate derivatives using the derivatizing reagents MSTFA and trimethylboroxine, respectively. For most of the compounds, lower detection limits were found for the TMS derivatives.

The alpha 2-adrenergic receptor system in the hypothalamus of the Pekin duck.

In the present study, we have employed the monoradioiodinated alpha 2-agonist clonidine ([125I]-CLO) to characterize duck hypothalamic alpha 2-adrenoceptors and to localize alpha 2-specific binding sites in the duck brain. To validate the alpha 2-specificity of [125I]-CLO using an enriched duck hypothalamic membrane fraction, a radioreceptor assay was established by altering the membrane protein concentration, time, temperature and ionic milieu of incubation, and in the presence or absence of protease inhibitors. Competitive displacement studies revealed the following sequence of potency to displace [125I]-CLO: yohimbine greater than (-)-epinephrine greater than clonidine greater than (-)-norepinephrine greater than phentolamine greater than (-)-phenylephrine greater than (-)-isoproterenol greater than prazosin. The non-hydrolyzable guanosine 5'-triphosphate analog guanylylimidodiphosphate markedly inhibited [125I]-CLO binding suggestive of G-protein involvement. With regard to the histological distribution, diencephalic structures, such as the habenula and the nucleus reticularis of the thalamus, were densely labeled by [125I]-CLO. In the hypothalamus, alpha 2-adrenoceptors were detected in the antidiuretic hormone-synthesizing nucleus paraventricularis, the nucleus praeopticus medialis, the nucleus anterior medialis hypothalami, the nucleus magnocellularis praeopticus, the nucleus commissurae pallii, the nucleus inferior hypothalami and the regio lateralis hypothalami. Circumventricular organs, such as the plexus choroidei, organum subfornicale, organum paraventriculare and the corpus pineale, were endowed with alpha 2-specific binding sites, as were the cell layers of the tectum opticum. In addition, telencephalic structures revealed high receptor densities.(ABSTRACT TRUNCATED AT 250 WORDS)

The metabolism of ICI 118,587, a partial agonist of beta 1-adrenoceptors, in mice, rats, rabbits, dogs, and humans.

The absorption, metabolism, and excretion of 14C-labeled xamoterol (ICI 118,587) has been examined in mice, rats, rabbits, dogs, and humans. There was incomplete absorption by all species after oral administration, ranging from 9% by humans to 36% by dogs. Most of the absorbed radioactivity was eliminated within 24 hr of administration and the renal route predominated. Conjugates of the parent compound were the only observed metabolites in urine, the phenolic glucuronide being the principal animal metabolite and the phenol sulfate being the only human metabolite. There were marked interspecies variations in metabolite patterns and dogs were the only animal species in which the sulfate metabolite was detected. Comparison of the urinary metabolite patterns also showed higher output of the conjugates after oral administration than after intravenous administration, indicating that first pass metabolism was taking place. Little significant change in absorption or metabolism was seen over a range of oral doses; in rats, some saturation of the glucuronide-conjugating mechanism was observed but the sulfate-conjugating mechanism showed little, if any, diminished capacity at high dose levels in dogs. The use of fast atom bombardment mass spectroscopy for the determination of the molecular weight of conjugates is described.