Co-localization of the alpha-subunit of BK-channels and c-PLA2 in GH3 cells.

Large conductance, calcium-activated potassium channels (maxi K- or BK-channels) can be regulated by arachidonic acid produced by c-Phospholipase A2 (c-PLA2). Since in every excised patch from GH3 cells where there was BK-channel activity, treatment with either a stimulator or inhibitor of c-PLA2 resulted in a corresponding increase or decrease in BK-channel activity, we hypothesized that there must be a close association between BK-channel proteins and c-PLA2 in the cell membrane. To test this hypothesis, we first determined whether the two proteins would co-immunoprecipitate. We then used confocal imaging of fluorescently tagged proteins to determine where in the cells BK-channel proteins and c-PLA2 co-localize. The alpha-subunit of the BK-channel was strongly co-immunoprecipitated by c-PLA2 antibodies, suggesting that most of the BK channel alpha-subunits are associated with c-PLA2. This interaction was not affected by pharmacologically inhibiting c-PLA2 suggesting that the association does not require functionally active c-PLA2. Following dual immunohistochemical labeling and confocal microscopy, image analysis revealed that in the cytosol there was some co-localization, but most of the c-PLA2 was separate from BK-channel proteins. On the other hand, the c-PLA2 and BK-channel proteins at the plasma membrane were strongly co-localized. Immunoprecipitation experiments conducted with plasma membrane proteins support these findings. We conclude that c-PLA2 is likely physically associated with BK-channel proteins at the cell surface.

Activation of BK channels in GH3 cells by a c-PLA2-dependent G-protein signaling pathway.

BK-channels in GH3 cells are activated by arachidonic acid produced by c-PLA2. beta-adrenergic agonists also activate BK channels and were presumed to do so via production of cAMP. We, however, show for the first time in GH3 cells that a beta-adrenergic agonist activates a pertussis-toxin-sensitive G protein that activates c-PLA2. The arachidonic acid produced by c-PLA2 then activates BK channels. We examined BK channels in cell-attached patches and in excised patches from untreated GH3 cells and from GH3 cells exposed to c-PLA2 antisense oligonucleotides. For the cell-attached patch experiments, physiologic pipette and bath solutions were used. For the excised patches, 150 mM KCl was used in both the pipette and bath solutions, and the cytosolic surface contained 1 microM free Ca2+ (buffered with 5 mM K2EGTA). Treatment of GH3 cells with the G protein activator, fluoroaluminate, (AlF4-) produced an increase in the Po of BK channels of 177 +/- 41% (mean +/- SD) in cell-attached patches. Because G proteins are membrane associated, we also added an activator of G proteins, 100 microM GTP-gamma-S, to the cytosolic surface of excised patches. This treatment leads to an increase in Po of 50 +/- 9%. Similar treatment of excised patches with GDP-beta-S had no effect on Po. Isoproterenol (1 microM), an activator of beta-adrenergic receptors and, consequently, some G proteins, increased BK channel activity 229 +/- 37% in cell-attached patches from cultured GH3 cells. Western blot analysis showed that GH3 cells have beta-adrenergic receptor protein and that isoproterenol acts through these receptors because the beta-adrenergic receptor antagonist, propanolol, blocks the action of isoproterenol. To test whether G protein activation of BK channels involves c-PLA2, we studied the effects of GTP-gamma-S on excised patches and isoproterenol on cell attached patches from GH3 cells previously treated with c-PLA2 antisense oligonucleotides or pharmacological inhibitors of c-PLA2. Neither isoproterenol nor GTP-gamma-S had any effect on Po in these patches. Similarly, neither isoproterenol nor GTP-gamma-S had any effect on Po in cultured GH3 cells pretreated with pertussis toxin. Isoproterenol also significantly increased the rate of arachidonic production in GH3 cells. These results show that some receptor-linked, pertussis-toxin-sensitive G protein in GH3 cells can activate c-PLA2 to increase the amount of arachidonic acid present and ultimately increase BK-channel activity.

Systemic tizanidine hydrochloride (Zanaflex) partially decreases experimental postoperative pain in rats.

IMPLICATIONS: Tizanidine was given systemically to rats with experimental postoperative pain. Partial pain relief was obtained, but the effect was not clinically significant. Although systemic tizanidine is effective in relieving experimental neuropathic pain, it is not as effective for postoperative pain.

Systemic tizanidine hydrochloride (Zanaflex) relieves thermal hyperalgesia in rats with an experimental mononeuropathy.

UNLABELLED: We sought to determine whether tizanidine, an alpha2-agonist, relieved thermal hyperalgesia in rats with surgically induced neuropathic pain. We used a Sprague-Dawley rat model in which a chronic constriction of the sciatic nerve caused the rats to develop postural changes, mechanical allodynia, and thermal hyperalgesia. Thermal hyperalgesia was verified through paw withdrawal latency (PWL). PWL was tested before surgery, after surgery, and after injections with tizanidine (0.5, 1.0, or 2.0 mg/kg) or normal saline. Ambulatory and total movements were evaluated by placing the rats in activity cages. Thermal hyperalgesia was induced in all rats after surgery. Tizanidine, but not saline, caused a significant improvement in PWL (P < 0.05), with complete reversal of thermal hyperalgesia at all doses on postoperative Day 6. Rats who received tizanidine 2 mg/kg maintained complete reversal of thermal hyperalgesia through postoperative Day 9. Some sedation was observed with tizanidine 2 mg/kg, but not with smaller doses. We conclude that tizanidine effectively reversed thermal hyperalgesia in a rat model. IMPLICATIONS: This study was conducted to determine whether tizanidine could attenuate the thermal hyperalgesia that occurs in rats with surgically induced chronic constriction of the sciatic nerve. Tizanidine was effective in reducing sensitivity to heat, as measured by paw withdrawal latency, and did not cause sedation at smaller doses.

Contrasting effects of cPLA2 on epithelial Na+ transport.

Activity of the epithelial Na+ channel (ENaC) is the limiting step for discretionary Na+ reabsorption in the cortical collecting duct. Xenopus laevis kidney A6 cells were used to investigate the effects of cytosolic phospholipase A2 (cPLA2) activity on Na+ transport. Application of aristolochic acid, a cPLA2 inhibitor, to the apical membrane of monolayers produced a decrease in apical [3H]arachidonic acid (AA) release and led to an approximate twofold increase in transepithelial Na+ current. Increased current was abolished by the nonmetabolized AA analog 5,8,11,14-eicosatetraynoic acid (ETYA), suggesting that AA, rather than one of its metabolic products, affected current. In single channel studies, ETYA produced a decrease in ENaC open probability. This suggests that cPLA2 is tonically active in A6 cells and that the end effect of liberated AA at the apical membrane is to reduce Na+ transport via actions on ENaC. In contrast, aristolochic acid applied basolaterally inhibited current, and the effect was not reversed by ETYA. Basolateral application of the cyclooxygenase inhibitor ibuprofen also inhibited current. Both effects were reversed by prostaglandin E2 (PGE2). This suggests that cPLA2 activity and free AA, which is metabolized to PGE2, are necessary to support transport. This study supports the fine-tuning of Na+ transport and reabsorption through the regulation of free AA and AA metabolism.

alpha-1 and alpha-2 Adrenergic antagonists relieve thermal hyperalgesia in experimental mononeuropathy from chronic constriction injury.

Phentolamine, a nonspecific alpha 1- and alpha 2-adrenergic antagonist, relieves pain in patients with reflex sympathetic dystrophy. We sought to determine whether phentolamine, prazosin (alpha 1 antagonist), or SKF86466 (alpha 2 antagonist) relieve thermal hyperalgesia in rats with neuropathic pain. Four days after producing a chronic constriction injury (CCI), thermal hyperalgesia was tested by measuring paw withdrawal latency (PWL). After injection of phentolamine, prazosin, or SKF86466 each at doses of 1, 2, or 5 mg/kg, PWL tests were measured at 5 min and repeated at 15-min intervals for 1 h. Phentolamine, prazosin, and SKF86466 1, 2, and 5 mg/kg provided statistically significant analgesia in rats with CCI for at least 65 min. PWL did not return to baseline levels after 1 or 2 mg/kg of prazosin or SKF86466 but did so after 35 min after phentolamine 2 mg/kg. After 5 mg/kg, PWL returned to preoperative values between 5 and 50 min for phentolamine, at 35 and 65 min for prazosin, and at 50 min for SKF86466. We conclude that both alpha1 and alpha2 peripheral receptors of the sympathetic nervous system are involved in the thermal hyperalgesia caused by CCI and that thermal hyperalgesia can be reversed by both alpha1 and alpha2 antagonists in a dose-dependent manner.

Results of immediate breast reconstruction after skin-sparing mastectomy.

Skin sparing mastectomy (SSM) removes the breast, nipple-areolar complex, previous biopsy incisions, and skin overlying superficial tumors. Preservation of the native skin envelope facilitates immediate breast reconstruction. The procedure has been adopted for the treatment of breast cancer. All cases of SSM and immediate breast reconstruction performed by the senior author (G.W.C.) from January 1, 1993, through December 12, 1997, were reviewed. Patient demographics, cancer staging, treatment, types of surgery performed, and postoperative outcomes were examined. Aesthetic outcomes were measured using four 3-point subscales. A total of 100 patients underwent 118 SSMs during the study period. The American Joint Committee on Cancer staging was as follows: stage 0, 27 patients; stage I, 25 patients; stage II, 39 patients; stage III, 7 patients; stage IV, 3 patients; recurrent, 2 patients; and cystosarcoma phylloides, 1 patient. The mean follow-up was 42.7 months. Local recurrence occurred in 2 patients (2.7%). Reconstructive methods included the transverse rectus abdominis musculocutaneous flap (N = 82; pedicled, 73; free, 9), the latissimus flap (N = 18), and tissue expansion (N = 20). Two patients underwent contralateral delayed reconstruction. The aesthetic results achievable with the three methods were similar. The failure rate was higher for expander reconstruction (10%) than those observed for transverse rectus abdominis musculocutaneous (4.9%) and latissimus (5.6%) flaps. SSM can be used in the treatment of invasive breast cancer without compromising local control. The aesthetic results of the three methods were similar, but tissue expander reconstruction had a higher failure rate.

Lack of pain associated with microfabricated microneedles.

Microscopic needles previously shown capable of transdermal delivery of drugs and proteins are demonstrated to be painless when pressed into the skin of human subjects.

Cytosolic phospholipase A2 is required for optimal ATP activation of BK channels in GH(3) cells.

To test the hypothesis that ATP activation of BK channels in GH(3) cells involves cytosolic phospholipase A(2) (cPLA(2)) as a potential protein target for phosphorylation, we first inhibited the activity of cPLA(2) by both pharmacologic and molecular biologic approaches. Both approaches resulted in a decrease rather than an increase in BK channel activity by ATP, suggesting that in the absence of cPLA(2), phosphorylation of other regulatory elements, possibly the BK channel protein itself, results in inactivation rather than activation of the channel. The absence of changes in activity in the presence of the non-substrate ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate verified that ATP hydrolysis was required for channel activation by ATP. Experiments with an activator and inhibitor of protein kinase C (PKC) support the hypothesis that PKC can be involved in the activation of BK channels by ATP; and in the absence of PKC, other kinases appear to phosphorylate additional elements in the regulatory pathway that reduce channel activity. Our data point to cPLA(2)-alpha (but not cPLA(2)-gamma) as one target protein for phosphorylation that is intimately associated with the BK channel protein.

Effects of fatty acids on BK channels in GH(3) cells.

Ca(2+)-activated K(+) (BK) channels in GH(3) cells are activated by arachidonic acid (AA). Because cytosolic phospholipase A(2) can produce other unsaturated free fatty acids (FFA), we examined the effects of FFA on BK channels in excised patches. Control recordings were made at several holding potentials. The desired FFA was added to the bath solution, and the voltage paradigm was repeated. AA increased the activity of BK channels by 3.6 +/- 1.6-fold. The cis FFA, palmitoleic, oleic, linoleic, linolenic, eicosapentaenoic, and the triple bond analog of AA, eicosatetraynoic acid, all increased BK channel activity, whereas stearic (saturated) or the trans isomers elaidic, linolelaidic, and linolenelaidic had no effect. The cis unsaturated FFA shifted the open probability vs. voltage relationships to the left without a change in slope, suggesting no change in the sensitivity of the voltage sensor. Measurements of membrane fluidity showed no correlation between the change of membrane fluidity and the change in BK channel activation. In addition, AA effects on BK channels were unaffected in the presence of N-acetylcysteine. Arachidonyl-CoA, a membrane impermeable analog of AA, activates channels when applied to the cytosolic surface of excised patches, suggesting an effect of FFAs from the cytosolic surface of BK channels. Our data imply a direct interaction between cis FFA and the BK channel protein.

Mechanisms of nonimmunological histamine and tryptase release from human cutaneous mast cells.

BACKGROUND: If mast cells are stimulated they release multiple mediators that delineate markers for immunologic and nonimmunologic reactions; histamine and tryptase are the two best known. Although histamine can be assayed in plasma, it is a nonspecific marker with a very short half-life. Tryptase has a longer half-life, but its release has not been proven to be specific for anaphylaxis. The authors investigated the mechanisms of nonimmunologic histamine release from human cutaneous mast cells to understand the mechanisms of mediator release and to determine whether tryptase was specific for allergic mediated activation. METHODS: Dispersed mast cell suspensions isolated from neonatal foreskins underwent challenge with vancomycin, calcium ionophore A23187, morphine, and atracurium, and histamine tryptase release was measured. The effects of calcium and magnesium, along with phospholipase C and phospholipase A2 inhibitors, also were investigated. RESULTS: Tryptase and histamine both were released by the known nonimmunologic stimuli (pharmacologic agents used in the current study; r2 = 0.6). Furthermore, vancomycin- and atracurium-induced histamine release was calcium dependent. Phospholipase C and phospholipase A2 inhibitors decreased vancomycin-induced histamine release, but not calcium ionophore A23187-induced release. CONCLUSIONS: Tryptase is not a specific marker of mast cell activation (ie., anaphylaxis), and signaling mechanisms for mast cell activation involve activation of phospholipase C and phospholipase A2 pathways that are also involved in other cellular activation mechanisms.

A comparison of the analgesic efficacy of 0.25% levobupivacaine combined with 0.005% morphine, 0.25% levobupivacaine alone, or 0.005% morphine alone for the management of postoperative pain in patients undergoing major abdominal surgery.

UNLABELLED: We compared the relative efficacy of the combination of the single-isomer local anesthetic levobupivacaine and the opioid analgesic morphine versus both drugs alone for postoperative epidural analgesia after major abdominal surgical procedures. Thoracic epidural anesthesia was produced and maintained with levobupivacaine 0.75% in combination with general inhaled anesthesia without opioids. Patients were randomized to one of three postoperative treatment groups: 1) a combination of levobupivacaine 0.25% and morphine 0.005%; 2) levobupivacaine 0.25%; or 3) morphine 0.005%. Postoperatively, all epidural infusions were commenced at a rate of 4 mL/h. Patients could receive a 4 mL-bolus dose and an increase in the epidural infusion rate by 2 mL/h on request for supplemental analgesia. Patients were also allowed ketorolac as a supplemental analgesic at any time after the first analgesic request. Patients in the combination group had longer times to request for supplemental analgesia as compared with the levobupivacaine only group (P < 0.05) and a trend toward longer time to request as compared with the morphine only group (P = 0.066). Patients in the combination group had lower visual analog scale pain scores at rest and activity at 4 and 8 h and fewer requests for supplemental ketorolac (P < 0.05). In conclusion, this study demonstrates a significant improvement in postoperative analgesic efficacy with the combination of levobupivacaine and morphine for continuous epidural analgesia after major abdominal surgical procedures. IMPLICATIONS: A significant improvement in postoperative analgesic efficacy is demonstrated with the thoracic epidural administration of the combination of the single-isomer local anesthetic levobupivacaine 0.25% and morphine 0.005% in patients after major abdominal surgical procedures as compared with either drug used alone.

Measurements of maternal protein binding of bupivacaine throughout pregnancy.

UNLABELLED: Pregnancy-related decreases in protein binding may contribute to altered effects of local anesthetics in the parturient. Previous studies have measured protein binding of bupivacaine in term parturients; the current study defines the ratio of bound-to-free bupivacaine throughout gestation at both therapeutic and toxic systemic concentrations of bupivacaine. Venous samples were obtained from 81 women, including 70 parturients, ranging from 7 to 42 wk of gestation and 11 nonpregnant controls. The percent bound bupivacaine at a fixed concentration was determined for each sample at both therapeutic (1 microg/mL) and toxic (5 microg/mL) concentrations using an ultrafiltration technique. Albumin and alpha-1-glycoprotein levels were also measured. Linear regression analysis showed a significant increase in concentration of free bupivacaine throughout gestation at the 5-microg/mL concentration, corresponding to a decrease demonstrated in both albumin and alpha-1-glycoprotein levels. A similar correlation was not found at the 1-microg/mL concentration. Although the relative magnitude of these changes is small, the relative change in free drug throughout gestation is large. Protein binding is only one of several mechanisms that may influence the susceptibility to local anesthetic toxicity in the parturient; however, its relative importance remains unclear. IMPLICATIONS: When venous samples taken from pregnant women were mixed with 5 microg/ml bupivacaine and analyzed, an increase in the free fraction of drug was seen with increasing gestational age, corresponding to decreases in alpha-1-glycoprotein and albumin.

Changes in rat paw perfusion after experimental mononeuropathy: assessment by laser Doppler fluxmetry.

UNLABELLED: This study was performed to determine the changes in perfusion that occur after chronic constriction injury (CCI). Male Sprague-Dawley rats weighing 275-300 g had loosely constricting ligatures placed around the left sciatic nerve. Paw withdrawal latency (PWL) to heat, skin temperature, and skin perfusion (laser Doppler) of the hind paws were measured before and for 30 days after CCI. PWL decreased significantly on the side of the CCI (maximum of 34% decrease on Postoperative Day [POD] 3), then returned to normal over a 20-day period. Skin temperature initially increased on the side of CCI, then decreased with respect to the control limb on PODs 20-30. Despite the initial increase in skin temperature on the side of CCI, skin perfusion significantly decreased immediately after CCI (maximum of 51% decrease on POD 6). The perfusion gradually returned to normal over 20 days. Because return to normal perfusion occurred while the skin temperature became colder than the control side, we conclude that there is no relationship between paw surface temperature and perfusion. IMPLICATIONS: Our data suggest that loss of sympathetic tone in thermoregulatory arteriovenous anastomoses leads to decreased nutritional blood flow to the skin of the affected limb after chronic constriction injury, which is consistent with the findings reported in humans with reflex sympathetic dystrophy.

Ca2+ sensitivity of BK channels in GH3 cells involves cytosolic phospholipase A2.

To test the hypothesis that intracellular Ca2+ activation of large-conductance Ca2+-activated K+ (BK) channels involves the cytosolic form of phospholipase A2 (cPLA2), we first inhibited the expression of cPLA2 by treating GH3 cells with antisense oligonucleotides directed at the two possible translation start sites on cPLA2. Western blot analysis and a biochemical assay of cPLA2 activity showed marked inhibition of the expression of cPLA2 in antisense-treated cells. We then examined the effects of intracellular Ca2+ concentration ([Ca2+]i) on single BK channels from these cells. Open channel probability (Po) for the cells exposed to cPLA2 antisense oligonucleotides in 0.1 microM intracellular Ca2+ was significantly lower than in untreated or sense oligonucleotide-treated cells, but the voltage sensitivity did not change (measured as the slope of the Po-voltage relationship). In fact, a 1,000-fold increase in [Ca2+]i from 0.1 to 100 microM did not significantly increase Po in these cells, whereas BK channels from cells in the other treatment groups showed a normal Po-[Ca2+]i response. Finally, we examined the effect of exogenous arachidonic acid on the Po of BK channels from antisense-treated cells. Although arachidonic acid did significantly increase Po, it did so without restoring the [Ca2+]i sensitivity observed in untreated cells. We conclude that although [Ca2+]i does impart some basal activity to BK channels in GH3 cells, the steep Po-[Ca2+]i relationship that is characteristic of these channels involves cPLA2.

A possible role for phospholipase A2 in the action of general anesthetics.

General anesthetics inhibit Ca(2+)-activated potassium (BK) channels at clinically relevant concentrations. This study examined the possibility that general anesthetics produce their effect on BK channels by disrupting the phospholipase A2 (PLA2)-arachidonic acid signal transduction pathway. Treatment of excised patches with exogenous arachidonic acid (2.5 microM) resulted in a 3.6 +/- 1.3-fold increase in BK channel activity. Subsequent exposure of these patches to concentrations of halothane (0.6 mM), ketamine (100 microM), or etomidate (10 microM) that would normally block the channel by approximately 60-80% in the absence of arachidonic acid did not reduce the channel activity. Arachidonic acid resulted in a significant increase in the 50% effective concentration for the ketamine dose-response curve from 3.4 +/- 0.4 to 693 +/- 379 microM (P < 0.001) as well as a significant decrease in slope from 1.40 +/- 0.21 to 0.59 +/- 0.05 (P < 0.001). The PLA2 inhibitors quinacrine (1 microM), aristolochic acid (250 microM), and octadecylbenzoylacrylic acid (7 microM) inhibited BK channels by 61 +/- 6, 47 +/- 2, and 30 +/- 9%, respectively, and in a manner indistinguishable from general anesthetics inhibition. Aristolochic acid and ketamine significantly inhibit the PLA2-mediated production of arachidonic acid in GH3 cells.

Ketamine inhibition of large conductance Ca(2+)-activated K+ channels is modulated by intracellular Ca2+.

The present investigation was conducted to study the relationship between intracellular Ca2+ and inhibition of large conductance Ca(2+)-activated K+ (BK) currents by ketamine using excised patches from GH3 cells. Five ketamine concentrations were studied in the presence of six Ca2+ concentrations. The half-maximal inhibition for BK channel block by ketamine was increased from 4.1 +/- 0.7 microM at 0.1 microM intracellular Ca2+ to 230 +/- 74 microM at 100 microM intracellular Ca2+. Open probability (Po), Ca2+ concentration, and ketamine concentration data were best described by a competitive inhibition model. The inhibition constant for ketamine was 20.5 +/- 5.2 microM, and the Michaelis-Menten constant (Km) value for Ca2+ was 3.58 +/- 0.49 microM, which was not different from Km for Ca2+ in the absence of ketamine (3.33 +/- 0.37 microM). Taken alone, these data would suggest that Ca2+ and ketamine were competing for the same site on the channel protein. However, examination of open and closed interval data from patches containing only one channel show that ketamine primarily produces a decrease in the frequency of long-lived open events, suggesting that the effect of ketamine on BK channels may not be by a direct effect on channel proteins.

Comparison of the efficacy of epidural morphine given by intermittent injection or continuous infusion for the management of postoperative pain.

BACKGROUND AND OBJECTIVES: To compare the effectiveness and side effects of epidural morphine sulfate (MSO4), delivered by continual infusion or intermittent bolus. METHODS: Thirty patients undergoing upper abdominal surgery were randomized into two equal groups to receive MSO4 through a thoracic epidural catheter by one of two methods. Group 1 patients received an initial bolus of morphine (0.07 mg/kg) at the end of surgery, followed by injections of 2-5 mg morphine into the epidural catheter on demand. Patients in group 2 received an initial bolus of morphine (0.03 mg/kg) during surgical peritoneal closure and were immediately started on an infusion of 0.01% morphine at 5 mL/hour (0.5 mg/hour). The infusion dose was titrated from 0.2 to 1.0 mg/hour, dependent on side effects. Outcome measurements included pulmonary function studies, arterial blood gases, morphine plasma levels, pain relief scores, global evaluations, and side effects. RESULTS: No difference existed between groups in forced vital capacity, forced expiratory volume in 1 second, or in arterial blood gas measurements. Side effects were similar in both groups. Respiratory depression was not seen in either group. Group 2 reported significantly better analgesia than group 1 on postoperative days 1 and 2 (P < .01). Peak plasma morphine levels for group 1 were significantly higher than the steady state plasma morphine levels for group 2 (P < .05). CONCLUSIONS: Continuous epidural infusion provides better analgesia without increased side effects for postoperative pain when compared with an intermittent (or demand) bolus technique.

The effect of racemic ketamine on the large conductance Ca(+2)-activated potassium (BK) channels in GH3 cells.

Recently, inhalation anesthetics have been reported to block BK channels in adrenal chromaffin cells. To determine if BK block was characteristic only of inhalation anesthetics or was also a property of other general anesthetics we examined the effects of ketamine, an intravenous general anesthetic which is structurally different than inhalation anesthetics. Cell-attached and excised patch single channel and standard whole cell recording techniques were used to examine the effect of racemic ketamine on the BK channel activity in GH3 cells. When solutions containing 150 mM KCl are used in both the pipette and bath, the BK channels are characterized as a voltage-dependent channel with a unit conductance of 150-300 pS. Racemic ketamine (at clinically relevant concentrations; 2-500 microM) selectively blocked BK channels in a dose-dependent, reversible manner as evidenced by decreases in NPo (number of channels x open probability). This decrease was due to both a decrease in mean open time and an increase in the mean closed time but without a decrease in single-channel current amplitude. Ketamine shifts the Po vs voltage curve to higher potentials without a change in the slope of the voltage dependence. Ketamine also shifts the Po vs [Ca+2] relationship to higher Ca+2 concentrations. The IC50 for the single-channel block by ketamine is 20.3 +/- 15.9 microM. In an effort to confirm that the effect of ketamine was predominantly due to a block of the BK channels, standard whole cell techniques were utilized.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics of intravenous and epidural ropivacaine in the rhesus monkey.

Ropivacaine is a new long-acting amide local anesthetic which is possibly less cardiotoxic than bupivacaine. The absorption and disposition of ropivacaine were characterized in six rhesus monkeys in an open two-way crossover study following intravenous and epidural administration. For these studies, animals were anesthetized for placement of intravenous and intraarterial catheters. For the epidural studies, a PE-10 catheter was also inserted 3 cm into the lumbar epidural space. After recovery from anesthesia, animals received ropivacaine 1 mg kg-1 intravenously over 1 min or 10 mg of ropivacaine epidurally (two 1 ml doses of 0.5%, 5 min apart), and arterial blood samples were obtained over 5 h. Serum ropivacaine concentrations were determined by gas chromatography with NP detection. Concentration-time data following i.v. and epidural administration were fitted simultaneously. Initial parameter estimates were obtained by analyzing each route separately. Input rates and their corresponding extent of absorption were estimated using deconvolution. Mean (+/- SD) disposition parameters included: Vss = 1.11 +/- 0.198 l kg-1; CL = 0.711 +/- 0.158 l h-1 kg-1; t1/2,z = 2.07 +/- 0.438 h. Mean (+/- SD) absorption parameters included: F1 = 0.506 +/- 0.221; t1/2,kal = 0.060 +/- 0.078 h; F2 = 0.444 +/- 0.182; t1/2,ka2 = 6.45 +/- 11.09 h. Ropivacaine's biphasic absorption and bioavailability are similar to those of other amide local anesthetics. The biphasic absorption may be related to partitioning into fat or regional changes in blood flow induced by the drug.