Post-tonsillectomy infiltration with bupivacaine reduces immediate postoperative pain in children.

Pain management after tonsillectomy in children remains a dilemma for the anaesthetist. A previous study demonstrated that the administration of lidocaine 1% topical spray to the peritonsillar fossae before tracheal extubation provided considerable immediate postoperative pain relief in infants and children. However, the pain relief was of short duration. We were hopeful that the use of bupivacaine would offer more prolonged pain relief because of its pharmacological characteristics. Therefore, this study was designed to compare the effects of bupivacaine 0.5% with 1:200,000 epinephrine administered after tonsillectomy either as topical spray or submucosal infiltration on postoperative pain in children. Forty-three patients aged two to ten years were randomized into three groups after tonsillectomy was performed. Group (1) received 0.5 ml.kg-1 normal saline spray; (2) received 2 mg.kg-1 bupivacaine 0.5% with 1:200,000 epinephrine peritonsillar infiltration in a similar volume to Group 1 and; (3) received 2 mg.kg-1 bupivacaine 0.5% with 1:200,000 epinephrine spray to both tonsillar beds. The patients in each group were compared postoperatively with regard to the quality of pain control using the Objective Pain Score, and their analgesic requirements. Peritonsillar infiltration of bupivacaine provided superior immediate postoperative analgesia as reflected by lower recovery room pain scores (P < 0.05) and opioid requirements (P < 0.01). Ward pain scores and analgesic requirements were similar among groups. Peritonsillar infiltration of bupivacaine 0.5% with 1:200,000 epinephrine provides better post-tonsillectomy pain control in the immediate postoperative period than bupivacaine spray or placebo.

An evaluation of the Kodak Ektachem clinical chemistry slide for theophylline.

We evaluated the Kodak Ektachem clinical chemistry slide for assay of theophylline. Assay precision and accuracy were acceptable in the therapeutic range although precision was poor at low levels of theophylline. The assay performed well with patients' samples using the Abbott TDx as the reference procedure but, as indicated by the manufacturer, uremic samples gave a positive bias, particularly in the therapeutic range. Finally, the significant bias observed with Quality Control material, probably due to matrix sensitivity, is a possible drawback.

Protein analysis of cardiac sarcolemma: effects of membrane-perturbing agents on membrane proteins and calcium transport.

Protein composition of cardiac sarcolemmal membranes was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Membranes were observed to contain about 20 polypeptide bands ranging from 18000 to 200 000 dalton mass. Out of these, six bands were prominent and together comprised 57% of the membrane protein. When sarcolemmal membranes, phosphorylated by [gamma-(32)P] ATP in the presence of Ca(2+) or Na+ with and without K+, were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at pH 2.4, the band III region (Mr 105 000) of gels was found to contain active sites of monomeric Ca-ATPase and (Na,K)ATPase. Bands I (Mr greater than 200 000), II (Mr 150 000), III (Mr 105 000), and VI (Mr 47 000) were accesible to trypsin; the extent of proteolysis was dependent on the time of exposure to, and the concentration of, trypsin (i.e, ratio of sarcolemmal protein/trypsin). Addition of molar sucrose protected sarcolemmal proteins from the tryptic proteolysis. Calcium transport was reduced by the action of trypsin; the degree of reduction was influenced by the time of exposure of membranes to trypsin as well as the concentration of trypsin. (Mg,Ca)ATPase activity, on the other hand, was elevated moderately at lower concentration and reduced at higher concentration of trypsin. Treatment with phospholipase C cium transport and (Mg,Ca)ATPase activity; electrophoretic patterns were unaffected by this treatment. Addition of lecithin to phospholipase C treated membranes produced a moderate increase in calcium transport. Exposure to Triton X-100 (1%) specifically solubilized three protein bands (Mr90 000, 67 000, and 57 000), whereas exposure to deoxycholate (1%) preferentially solubilized high-molecular-weight proteins, including band III (Mr 105 000); Lubrol-PX (1%) caused nonspecific solubilization of proteins, although the extent of solubilization with Lubrol-PX was considerably less than with either Triton or deoxycholate.

Stimulation of cardiac sarcolemmal (Na+--K+) ATPase activity by phosphorylase kinase.

Following preincubation with phosphorylase kinase, ATPase activities of heart sarcolemmal membranes were increased: total ATPase from 9.38+/-0.65 to 15.25+/-0.90 and ouabain-sensitive (Na+--K+)ATPase from 1.67+/-0.17 to 3.12+/-0.33 micron moles Pi/mg protein/h (mean +/- S.E. of 3 experiments); (Ca2+)ATPase and (Mg2+--Ca2+)-ATPase activities were not significantly altered due to phosphorylase kinase. Under these conditions, phosphorylase kinase catalyzed phosphorylation of sarcolemmal membranes. The kinase-catalyzed phosphorylation of membranes was increased by Ca2+ ions: at pH 6.8, 30% increase in phosphorylation was observed whereas at pH 8.5, 267% increase was noted due to this action. These findings support the view that Ca2+-dependent phosphorylation of membranes regulates (Na+--K+)ATPase.

Stimulation of calcium accumulation in cardiac sarcolemma by phosphorylase kinase.

After incubation with phosphorylase kinase, calcium accumulation in cardiac sarcolemma was increased from 65+/-2 to 95+/-3nmol/10min per mg of protein. Under these conditions, phosphorylase kinase catalysed phosphorylation of membranes. This phosphorylation was hydroxylamine-insensitive, was stimulated by Ca2+ ions and was unaffected by 3':5'-cyclic AMP.

Adenosine triphosphate - dependent calcium binding and accumulation by guinea pig cardiac sarcolemma.

Sarcolemma isolated from guinea pig heart ventricles possessed ATP-dependent Ca2+ binding and accumulation (+ oxalate) activities which were not inhibited by sodium azide, oligomycin, or ruthenium red. Ca2+ binding and accumulation by sarcolemma were sensitive to pH, the optimum being about pH 6.8. The concentrations of ATP required for half-maximal binding and accumulation were 94.3 and 172 muM, respectively. Mg2+ up to 5 mM significantly enhanced both activities but was inhibitory at higher concentrations (greater than 10 mM). Sarcolemmal Ca2+ binding and accumulation were stimulated 100% by K+, half-maximal enhancement occurring at 5-10 mM K+. Ca2+ binding and accumulation were both saturable processes and the respective apparent Km values for Ca2+ were 16.4 and 14.3 muM. Ca2+ binding by sarcolemma was a rapid process and the bound Ca2+ was released upon depletion of ATP in the medium. It is suggested that the sarcolemmal Ca2+ transport system may well be of significance in regulation of the contraction-relaxation cycle of cardiac muscle.

Guanylate cyclase. Subcellular distribution in cardiac muscle, skeletal muscle, cerebral cortex and liver.

1. Guanylate cyclase of every fraction studied showed an absolute requirement for Mn2+ ions for optimal activity; with Mg2+ or Ca2+ reaction was barely detectable. Triton X-100 stimulated the particulate enzyme much more than the supernatant enzyme and solubilized the particulate-enzyme activity. 2. Substantial amounts of guanylate cyclase were recovered with the washed particulate fractions of cardiac muscle (63-98%), skeletal muscle (77-93%), cerebral cortex (62-88%) and liver (60-75%) of various species. The supernatants of these tissues contained 7-38% of total activities. In frog heart, the bulk of guanylate cyclase was present in the supernatant fluid. 3. Plasma-membrane fractions contained 26, 21, 22 and 40% respectively of the total homogenate guanylate cyclase activities present in skeletal muscle (rabbit), cardiac muscle (guinea pig), liver (rat) and cerebral cortex (rat). In each case, the specific activity of this enzyme in plasma membranes showed a five- to ten-fold enrichment when compared with homogenate specific activity. 4. These results suggest that guanylate cyclase, like adenylate cyclase, and ouabain-sensitive Na+ + K+-dependent ATPase (adenosine triphosphatase), is associated with the surface membranes of cardiac muscle, skeletal muscle, liver and cerebral cortex; however, considerable activities are also present in the supernatant fractions of these tissues which contain very little adenylate cyclase or ouabain-sensitive Na+ + K+-dependent ATPase activities.

Adenylate cyclase, guanylate cyclase and cyclic nucleotide phosphodiesterases of guinea-pig cardiac sarcolemma.

1. The activities of the enzymes involved in the metabolism of cyclic nucleotides were studied in sarcolemma prepared front guinea-pig heart ventricle; the enzyme activities reported here were linear under the assay conditions. 2. Adenylate cyclase was maximally activated by 3mM-NaF; NaF increased the Km for ATP (from 0.042 to 0.19 mM) but decreased the Ka for Mg2+ (from 2.33 to 0.9 mM). In the presence of saturating Mg2+ (15 mM), Mn2+ enhanced adenylate cyclase, whereas Co2+ was inhibitory. beta-Adrenergic amines (10-50 muM) stimulated adenylate cyclase (38+/-2%). When added to the assay mixture, guanyl nucleotides (GTP and its analogue, guanylyl imidophosphate) stimulated basal enzyme activity and enhanced the stimulation by isoproterenol. By contrast, preincubation of sarcolemma with guanylyl imidodiphosphate stimulated the formation of an 'activated' form of the enzyme, which did not reveal increased hormonal sensitivity. 3. The guanylate cyclase present in the membranes as well as in the Triton X-100-solubilized extract of membranes exhibited a Ka for Mn 2+ of 0.3 mM; Mn2+ in excess of GTP was required for maximal activity. Solubilized guanylate cyclase was activated by Mg2+ only in the presence of low Mn2+ concentrations; Ca2+ was inhibitory both in the absence and presence of low Mn2+. Acetylcholine as well as carbamolycholine stimulated membrane-bound guanylate cyclase. 4. Cylic nucleotide phosphodiesterase activities of sarcolemma exhibited both high-and low-Km forms with cyclic AMP and with cyclic GMP as substrate. Ca2+ ions increased the Vmax. of the cyclic GMP-dependent enzyme.

Isolation and enzymatic characterization of guinea pig cardiac sarcolemma.

Sarcolemma was isolated by fractionation of salt-extracted particles on two consecutive sucrose density gradients. Salt extraction of homogenates, rather than of washed particles, was found to preserve the activities of adenylate cyclase and ouabain-sensitive (Na+,-K+)-ATPase in the isolated sarcolemmal membranes. Purified sarcolemma contained substantial adenylate cyclase and guanylate cyclase activities that were stimulable by beta-adrenergic and muscarinic agonists, respectively. Significant ouabain-sensitive (Na+, K+)-ATPase activity as well as putative digitalis receptor activity was also present in sarcolemma. Cyclic nucleotide phosphodiesterases of sarcolemma, both cAMP- and cGMP-dependent, displayed positive cooperativity of substrate interactions; Ca2+ ions were found to increase the activity of the GMP-dependent enzyme.

Characteristics of heart sarcolemmal calcium transport system and effect of protein kinase on sarcolemmal calcium accumulation.

Properties of the ATP-dependent calcium transport system of heart sarcolemma are presented. Calcium accumulation (with oxalate) in sarcolemma was increased due to cAMP-dependent protein kinase and phosphorylase b kinase. Protein kinase increased the Vmax of the sarcolemmal calcium accumulation without any detectable effect on the affinity for Ca2+. Both kinases failed to stimulate calcium binding. Protein kinase catalyzed phosphorylation of membrane proteins of molecular weights of 100,000, 25,000, and 14,000. Phosphorylase b kinase also catalyzed phosphorylation of these proteins. Protein kinase stimulated ATPase activity of sarcolemma. Sarcolemma contained endogenous protein kinase and protein phosphatase activities.

Stimulation of calcium accumulation in cardiac sarcolemma by protein kinase.

Sarcolemmal membranes isolated from guinea pig heart ventricles contained an ATP-dependent calcium-sequestering activity. Sarcolemmal calcium accumulation but not binding was enhanced by preincubation of membranes with exogenous protein kinase, with cyclic AMP, or with isoproterenol. Protein kinase (EC 2.7.1.37) increased the V of Ca2+ accumulation by sarcolemma without any significant effect on the affinity for Ca2+. The endogenous protein kinase activity present in isolated sarcolemma affected membrane phosphorylation. Cyclic AMP increased the endogenous kinase activity modestly, whereas histone increased it significantly. Exogenous protein kinase also catalyzed phosphorylation of these membranes. Endogenous and exogenous kinase-catalyzed phosphorylation of sarcolemma was hydroxylamine-insensitive. Ca2+-dependent ATPase (EC 3.6.1.3) (extra ATPase) activity of sarcolemma was also increased by protein kinase.