Studies on the mechanism of [3H]-noradrenaline release from SH-SY5Y cells: the role of Ca2+ and cyclic AMP.

1. The roles of both Ca2+ and adenosine 3':5'-cyclic monophosphate (cyclic AMP) in carbachol and K(+)-stimulated [3H]-noradrenaline release from SH-SY5Y human neuroblastoma cells were examined. 2. Both carbachol and K+ caused a time- and dose-related stimulation of [3H]-noradrenaline release. The release event in perfused cells was monophasic. Half-maximum stimulation measured in statically incubated (3 min) cells was 38 +/- 4 microM and 63 +/- 4 mM respectively. K+ (100 mM, added)-evoked release was greater than that produced by carbachol (1 mM). 3. Both carbachol and K+ caused a time- and dose (measured at 3 min)-related stimulation of cyclic AMP formation with half-maximum stimulation occurring at 5 +/- 1 microM and 49 +/- 2 mM respectively. In contrast to its effects on release, carbachol produced a greater stimulation of cyclic AMP formation than K+. 4. K(+)-stimulated [3H]-noradrenaline release was entirely dependent on Ca2+ entry as 2.5 mM Ni2+ abolished release. However, carbachol-evoked (1 mM) release appeared to be unaffected by Ni2+ pretreatment. 5. These data suggest that in SH-SY5Y cells, elevated cyclic AMP levels are not directly involved in [3H]-noradrenaline release. In addition, carbachol-stimulated release is largely independent of extracellular Ca2+ possibly implying a role for intracellular stored Ca2+ in the release process.

Effects of morphine and its metabolites on opiate receptor binding, cAMP formation and [3H]noradrenaline release from SH-SY5Y cells.

Opiate receptor occupation leads to a variety of intracellular events including inhibition of adenylyl cyclase and cAMP formation. We have examined the opiate binding characteristics, effects on cAMP formation and [3H]noradrenaline release of morphine, morphine-6 (M6G) and -3 (M3G)-glucuronides, and fentanyl in SH-SY5Y human neuroblastoma cells. M6G and M3G are the major metabolites of morphine formed in vivo whose cellular action remains to be fully elucidated. In binding experiments morphine (affinity, K50 = 96 nM) and fentanyl (K50 = 99 nM) were more potent than M6G (K50 = 393 nM), while M3G was inactive. However, for cAMP inhibition morphine (half maximum inhibition, IC50 = 193 nM) and M6G (IC50 = 113 nM) were roughly equipotent, with fentanyl (IC50 = 27 nM) being more potent and producing a greater maximum inhibition (56%). M3G was inactive. These in vitro data are in general agreement with the in vivo effects of these glucuronides. Moreover, all of the opiates tested failed to inhibit K(+)-evoked release of [3H]noradrenaline. Whilst these data do not support a role for cAMP in neurotransmitter release, alterations in cAMP formation may still have a role to play in the mechanism of analgesia.

Effects of propofol and thiopentone on potassium- and carbachol-evoked [3H]noradrenaline release and increased [Ca2+]i from SH-SY5Y human neuroblastoma cells.

We have examined the effects of two intravenous anaesthetic induction agents, propofol and thiopentone, on K+ and carbachol evoked [3H]noradrenaline release from a human neuroblastoma cell line, SH-SY5Y. In this model, we have previously demonstrated that K+ evoked [3H]noradrenaline release was dependent on Ca2+ entry and carbachol evoked release was extracellular Ca(2+)- independent. Propofol inhibited K+ (100 mM)-evoked (IC50 of 42 +/- 11 microM), but not carbachol (1 mM)-evoked, [3H]noradrenaline release. Thiopentone inhibited both K+- and carbachol-evoked release with IC50 values of 116 +/- 15 microM and 169 +/- 39 microM, respectively. These inhibitory effects were not due to changes in the release dynamics, as assessed using perfused cells. Furthermore, thiopentone inhibition of carbachol-evoked release was not due to muscarinic receptor antagonism. Both propofol and thiopentone caused noncompetitive inhibition of K+-stimulated Ca2+ influx, with IC50 values of 127 +/- 7 microM and 121 +/- 10 microM, respectively. These effects were not due to interaction with GABAA receptors, but suggest that both compounds block voltage-sensitive Ca2+ channels. Thiopentone, but not propofol, inhibited carbachol-stimulated increased intracellular Ca2+ concentrations in the presence and absence of extracellular Ca2+. However, thiopentone had no effect on carbachol-stimulated inositol (1,4,5)-triphosphate formation, suggesting that thiopentone may directly inhibit Ca2+ release from intracellular stores.

Interaction of local anaesthetic agents with the endogenous norepinephrine transporter in SH-SY5Y human neuroblastoma cells.

Use of intravenous guanethidine for the treatment of complex regional pain syndrome type I is of variable efficacy. Guanethidine injection is painful, so local anaesthetic is co-administered. We hypothesize that local anaesthetic inhibits uptake of guanethidine and hence reduces its efficacy. In this study we have examined the effects of a range of local anaesthetic agents on the uptake of [3H]norepinephrine ([3H]NE) (as a surrogate for guanethidine) and the binding of [3H]nisoxetine to the NE transporter in cultured SH-SY5Y human neuroblastoma cells. All local anaesthetic agents inhibited NE uptake with a rank order cocaine>tetracaine>procaine(esters), dibucaine > bupivacaine > prilocaine > lidocaine (amides). In addition all anaesthetic agents displaced [3H]nisoxetine with a rank order cocaine > tetracaine > dibucaine > procaine > prilocaine > bupivacaine > lidocaine. There was a positive correlation between [3H]NE uptake and [3H]nisoxetine binding. Our data suggest that when local anaesthetic and guanethidine are co-administered the former may reduce uptake of the latter and hence reduce the clinical efficacy of guanethidine.

Evaluation of protocols allowing emergency medical technicians to determine need for treatment and transport.

OBJECTIVE: To determine whether emergency medical technicians (EMTs) can safely apply protocols to assign transport options and to assess agreement between groups of providers on application of the protocols. METHODS: Developed protocols categorized patients as: 1) needs ambulance; 2) go to the emergency department (ED) by alternative means; 3) contact primary care provider (PCP); or 4) treat and release. After education on the application of the protocols, first responders and ambulance EMTs categorized patients at the scene prior to transport but did not change current practice. Ambulance reports were reviewed using a predetermined list of critical events that signified the need for an ambulance. RESULTS: The EMTs categorized 1,300 study patients as follows: 1,023 (79%) needed ambulance transport, 200 (15%) could go to the ED by alternative means, 63 (5%) could contact a PCP, 14 (1%) could be treated and released. Categorizations by a first responder and the transporting EMT were compared for 209 patients. Collapsing categories to "need ambulance/do not need ambulance" showed fair concordance (kappa = 0.51). Initially, 30 of 277 (11%) patients determined not to need an ambulance appeared to experience a critical event. After review, 23 patients had events that may not warrant advanced life support transport. Seven (3%) had critical events in the ambulance warranting ambulance transport. Most were miscategorized by the EMT. Overall sensitivity and specificity for identifying patients needing ambulance transport were 94.5% and 32.8%, respectively. CONCLUSIONS: From 3% to 11% of patients determined on scene not to need an ambulance had a critical event. Emergency medical services systems need to determine an acceptable rate of undertriage. Further study is needed to determine whether better adherence to the protocols might increase safety.

Do patients refusing transport remember descriptions of risks after initial advanced life support assessment?

OBJECTIVE: To determine patient recall and understanding of instructions given to patients who refuse transport after initial paramedic assessment and medical treatment. METHODS: Following patient consent, a phone interview was completed for consecutive persons living in a large urban area for whom 9-1-1 was contacted but who subsequently refused transport after advanced life support (ALS) assessment. Subjects were asked about their recall of explained risks and benefits of transport, their understanding of those risks at the time of assessment, and subsequent use of medical care, including hospitalization. RESULTS: From October 1, 1996, to February 23, 1997, 324 people refused transport after ALS arrival. Sixty-eight people could not be contacted, providing a response rate of 79% (256/324). Six percent were subsequently admitted to the hospital for the same problem and an additional 59% sought care from a health care provider (66 ED visits, 63 personal physician, 16 urgent care, 5 other). There were no unexpected deaths. Ninety (35%) respondents were still experiencing symptoms at the time of phone contact. Despite the routine practice of providing a verbal explanation of risks and written instructions, only 141 (55%) recalled receiving written instructions and 56 (22%) recalled an explanation of risks. Twenty-six percent believed they did not fully understand their conditions or circumstances surrounding the 9-1-1 call when they refused transport and 18% would now take an ambulance if the same incident were to recur. CONCLUSION: A substantial proportion of patients refusing transport do not recall receiving verbal or written instructions and would reconsider their transport decision, raising doubts about people's ability to make informed decisions at a time of great vulnerability. The majority of patients accessed health care after refusing transport and 6% were hospitalized.

Fentanyl inhibits the release of [3H]noradrenaline from SH-SY5Y human neuroblastoma cells.

We have examined the effect of fentanyl on [3H]noradrenaline release in a human neuroblastoma cell preparation, SH-SY5Y. Fentanyl produced a significant, concentration-dependent inhibition of [3H]noradrenaline release with IC50 values of 5.5 x 10(-6) mol litre-1 and 15.5 x 10(-6) mol litre-1 for carbachol- and potassium-evoked release, respectively. The small difference in IC50 between the two evoking stimuli may be explained by the weak binding affinity of fentanyl to muscarinic receptors (Ki = 570 nmol litre-1). The minimum concentrations at which a significant effect was observed were 0.3 x 0.10(-6) mol litre-1 and 10.0 x 10(-6) mol litre-1 for carbachol- and potassium-evoked release, respectively; these values are considerably in excess of the serum concentration of fentanyl required to produce analgesia. Naloxone failed to antagonize the fentanyl inhibition and, furthermore, morphine and an enkephalin had no effect on evoked release, implying a non-opioid receptor mediated effect.

Fentanyl inhibits the uptake of [3H]noradrenaline in cultured neuronal cells.

We have examined how fentanyl modulates [3H]noradrenaline uptake in two cultured neuronal cell preparations, the human neuroblastoma SH-SY5Y and the rat phaeochromocytoma PC12. Fentanyl produced a significant, dose-dependent inhibition of [3H]noradrenaline uptake at concentrations in excess of 0.1 mumol litre-1 (P < 0.05) and 0.3 mumol litre-1 (P < 0.05) for PC12 and SH-SY5Y cells, respectively. However, these values exceed the serum concentration of fentanyl required to produce analgesia. At the maximum concentration examined (100 mumol litre-1), fentanyl produced 85-95% inhibition of uptake. This effect was not antagonized by naloxone, implying a nonopioid mechanism of action. Imipramine 1 mumol litre-1 reduced [3H]noradrenaline uptake by 65-70% but morphine, in contrast to fentanyl, had no effect (P > 0.1).

Pre-emptive analgesia with local anaesthetic for herniorrhaphy.

There is little evidence that local anaesthetics produce pre-emptive analgesia and one reason may be the short duration of action of the drugs studied. We examined the pre-emptive analgesic effect of a bupivacaine field block on postoperative pain in 40 patients following herniorrhaphy in a double-blind, randomised trial. Patients received the block either after induction but before surgery, or after surgery but before the end of anaesthesia. There was no difference in pain scores or analgesic consumption up to 7 days after surgery between the two groups. We have demonstrated that bupivacaine does not appear to provide significant pre-emptive analgesia following a field block for herniorrhaphy. This study does not support the hypothesis that pre-emptive analgesia with local anaesthetic depends upon the duration of action of the drug.

Effect of halothane on K+ and carbachol stimulated [3H]noradrenaline release and increased [Ca2+]i in SH-SY5Y human neuroblastoma cells.

We have examined the effects of the volatile general anaesthetic agent, halothane, on K+ and carbachol stimulated [3H]noradrenaline release and associated increases in intracellular Ca2+ in a cultured human neuroblastoma cell line, SH-SY5Y. K+ (but not carbachol) stimulated [3H]noradrenaline release, and the increase in intracellular Ca2+ concentration was entirely extracellular Ca2+ dependent. Halothane produced a dose-dependent reduction in K+ evoked release of [3H]noradrenaline with significant inhibition (17%) occurring from 1.26 atm%. Basal and carbachol evoked release were unaffected. Halothane also produced a dose-dependent reduction in K+ evoked increases (measured at the peak) in intracellular Ca2+ with significant inhibition (29%) occurring from 0.88 atm%. K+ plateau, basal and carbachol evoked increases in intracellular Ca2+ were unaffected. These data suggest that halothane reduced Ca2+ entry through voltage-sensitive Ca2+ channels and implicate this important class of ion channel in the mechanism of anaesthesia.

Hospital follow-up of patients categorized as not needing an ambulance using a set of emergency medical technician protocols.

OBJECTIVE: Using hospital outcomes, this study evaluated emergency medical technicians' (EMTs') ability to safely apply protocols to assign transport options. METHODS: Protocols were developed that categorized patients as: 1) needs ambulance; 2) may go to emergency department (ED) by alternative means; 3) contact primary care provider (PCP); or 4) treat and release. After education on application of the protocols, EMTs categorized patients at the scene prior to transport but did not change current practice. Hospital charts were reviewed to determine outcome of patients whom EMTs categorized as not needing an ambulance. Category 2 patients were assumed to need the ambulance if they were admitted to a monitored bed or intensive care unit. Category 3 and 4 patients were assumed to need the ED if they were admitted. RESULTS: The EMTs categorized 1,300 study patients: 1,023 (79%) ambulance transport, 200 (15%) alternative means, 63 (5%) contact PCP, and 14 (1%) treat and release. Hospital data were obtained for 140 (51%) patients categorized as not needing ambulance transport. Thirteen of 140 (9%) patients who transporting EMTs determined did not need the ambulance were considered to be undertriaged: five in category 2, six in category 3, and one in category 4. Six of 13 (46%) undertriaged patients had dementia or a psychiatric disorder as one of their presenting complaints. CONCLUSION: These protocols led to a 9% undertriage rate. Patients with psychiatric complaints and dementia were at high risk for undertriage.

Using the Sustained Attention to Response Test to detect cognitive dysfunction after day case surgery.

BACKGROUND AND OBJECTIVE: Cognitive dysfunction has been reported after general anaesthesia, but its assessment is time consuming and difficult to evaluate. This pilot study assessed the feasibility of using the Sustained Attention to Response Test to assess 35 ASA I-II adults (mean age 31.6 yr) undergoing day case surgery under general anaesthesia, and 25 ASA I-II adults (mean age 47.8 yr) undergoing day case surgery under local anaesthesia. METHODS: The Sustained Attention to Response Test was performed before surgery and repeated 2 h after surgery. RESULTS: When patients repeated the test after surgery under local anaesthesia, the number of incorrect responses increased, but reaction times decreased (P < 0.05). Following general anaesthesia, the number of incorrect responses increased (P < 0.05), but reaction times remained unchanged. CONCLUSIONS: The Sustained Attention to Response Test is simple to administer and may be a useful tool when comparing different anaesthetic techniques and their effects on postoperative deficits in sustained attention.