Use of ROC curve analysis for prediction gives fallacious results: Use predictivity-based indices.

ABSTRACT: The area under the ROC curve is frequently used for assessing the predictive efficacy of a model, and the Youden index is commonly used to provide the optimal cut-off. Both are misleading tools for predictions. A ROC curve is drawn for the sensitivity of a quantitative test against its (1 - specificity) at different values of the test. Both sensitivity and specificity are retrospective in nature as these are indicators of correct classification of already known conditions. They are not indicators of future events and are not valid for predictions. Predictivity intimately depends on the prevalence which may be ignored by sensitivity and specificity. We explain this fallacy in detail and illustrate with several examples that the actual predictivity could differ greatly from the ROC curve-based predictivity reported by many authors. The predictive efficacy of a test or a model is best assessed by the percentage correctly predicted in a prospective framework. We propose predictivity-based ROC curves as tools for providing predictivities at varying prevalence in different populations. For optimal cut-off for prediction, in place of the Youden index, we propose a P-index where the sum of positive and negative predictivities is maximum after subtracting 1. To conclude, for correctly assessing adequacy of a prediction models, predictivity-based ROC curves should be used instead of the usual sensitivity-specificity-based ROC curves and the P-index should replace the Youden index.

A randomised controlled trial of phenylephrine and noradrenaline boluses for treatment of postspinal hypotension during elective caesarean section.

Phenylephrine is currently the vasopressor of choice during elective caesarean section, but it can cause reflex bradycardia. Noradrenaline, a potent α-agonist and weak ß-agonist, may be associated with a lower incidence of bradycardia. However, comparative information is limited. This double-blind randomised controlled trial compared the effects of 100 µg phenylephrine and 5 µg noradrenaline administered as boluses for the treatment of postspinal hypotension during elective caesarean section in women with an uncomplicated singleton pregnancy. Hypotension was defined as a decrease of ≥ 20% from baseline systolic arterial pressure, or an absolute value < 100 mmHg. Ninety women were included in the study. The primary outcome was the incidence of maternal bradycardia < 60 beats.min-1 . There was no difference in the incidence of bradycardia (37.8% with phenylephrine vs. 22.2% with noradrenaline; p = 0.167), number of hypotensive episodes, number of boluses required to treat the first hypotensive episode or reactive hypertension. The total number of boluses used was higher in the phenylephrine group (p = 0.01). Maternal heart rate at 1 min after vasopressor administration was non-significantly lower using phenylephrine vs. noradrenaline (p = 0.034, considering p < 0.01 as statistically significant). The umbilical artery pH was higher using phenylephrine than with noradrenaline (p = 0.034). In conclusion, both vasopressors reversed postspinal hypotension without a statistically significant difference in maternal bradycardia. However, in view of the lower umbilical artery pH when using noradrenaline, further research is warranted to study its placental transfer and fetal metabolic effects.

The minimum effective dose (ED90) of prophylactic oxytocin infusion during cesarean delivery in patients with and without obesity: an up-down sequential allocation dose-response study.

BACKGROUND: Obesity is associated with greater oxytocin requirement during labor induction or augmentation. There are scant data exploring the intra-operative requirement during cesarean delivery in patients with obesity, and none comparing it with those without obesity. We evaluated the minimum effective dose (ED90) of an oxytocin infusion to achieve adequate uterine tone during cesarean delivery in patients with and without obesity. METHODS: Patients (body mass index ≥30 kg/m2 represented patients with obesity) undergoing cesarean delivery using subarachnoid block were included. This prospective dual-arm dose-finding study used a 9:1 biased sequential allocation design. Oxytocin infusion was initiated at 13 IU/h at cord clamping in the first patient of each group. Uterine tone was graded as satisfactory or unsatisfactory by the obstetrician four minutes after initiation of the infusion. The dose of oxytocin infusion for subsequent patients was determined according to the response of the previous patient in the group. Oxytocin-associated side effects were evaluated. Dose-response data for the groups was evaluated using log-logistic function and ED90 estimates derived from fitted equations using the delta method. RESULTS: The ED90 of oxytocin was significantly higher for patients with obesity (n = 40) compared with those without obesity (n = 40) [25.7 IU/h, 95% CI 18.6 to 32.9) vs. 16.6 IU/h, 95% CI 14.9 to 18.3)]; relative ratio 1.55 [95% CI 1.09 to 2.01] (P = 0.019). CONCLUSIONS: Patients with obesity require a higher intra-operative oxytocin infusion dose rate to achieve a satisfactorily contracted uterus after fetal delivery when compared with patients without obesity.

Environment-friendly route for stable aqueous dispersion of reduced graphene oxide for heat transfer application.

A simultaneous functionalization and reduction route to prepare stable dispersion of reduced graphene oxide from graphene oxide has been described. Diethanol amine has been introduced for the first time as an environment friendly reducing agent in a simple reflux reaction. Diethanol amine acts as a reducing agent and helps to enhance the stability of dispersion, making hydrogen bonding by virtue of two functional groups. The prepared dispersion of 0.025 mg/mL concentration is stable for months together and has a zeta potential value -45 V at room temperature. UV-Vis study shows peak at 264 nm that is signatory for reduced graphene oxide. TEM images confirm spread thin sheets of graphene of few hundred nanometer lateral dimension. Thermal diffusivity studies suggest nearly 60% enhancement for the dispersion in comparison to base fluid, water. This suggests graphene dispersion is promising for heat transfer applications.

Calculation of effective dose of phenylephrine bolus for treatment of post-spinal hypotension in pre-eclamptic patients undergoing caesarean section - a non-randomised controlled trial.

BACKGROUND: Patients with pre-eclampsia require smaller vasopressor doses compared with those with normotension for management of post-spinal hypotension during caesarean section. However, the literature has little evidence as to the phenylephrine dose required for patients with pre-eclampsia. METHODS: Fifty patients, with either pre-eclampsia or normotension, and developing post-spinal hypotension during caesarean section under spinal anaesthesia, were studied. Women in both groups did not receive prophylactic vasopressors. The first patient in each group received phenylephrine 50 µg to treat the first episode of hypotension, defined as fall of systolic blood pressure ≥20% from baseline or an absolute value <100 mmHg. If hypotension was corrected within one minute it was considered a 'success'. The doses for the subsequent patients were determined by responses to all previous patients, according to a variation of Narayana's rule for the up-down sequential allocation method. RESULTS: The 95% effective dose (ED95) and 50% effective dose (ED50) of phenylephrine was 41.7 µg (95% CI 33.8 to 49.6 µg) and 29.1 µg (95% CI 26.0 to 32.2 µg) respectively in the pre-eclampsia group, and 64.9 µg (95% CI 54.1 to 75.7 µg) and 47.3 µg (95% CI 39.7 to 54.9 µg) respectively in the normotensive group. The proportionate reduction in phenylephrine dose ranged from 33% (95% CI 18 to 44%) to 40% (95% CI 19 to 52%). CONCLUSION: Patients with pre-eclampsia may need a 33% to 40% reduction in the first phenylephrine bolus dose, compared with patients with normotension, for the treatment of the first episode of post-spinal hypotension.

Neonatal outcomes following phenylephrine or norepinephrine for treatment of spinal anaesthesia-induced hypotension at emergency caesarean section in women with fetal compromise: a randomised controlled study.

BACKGROUND: Norepinephrine is as effective as phenylephrine for management of spinal anaesthesia-induced hypotension. Most of the studies comparing these vasopressors have been conducted in healthy pregnant women undergoing elective caesarean section. In the current study, we tested the null hypothesis that there is no difference in neonatal outcome when phenylephrine or norepinephrine is used to treat spinal anaesthesia-induced hypotension in women undergoing emergency caesarean section for fetal compromise. METHODS: Patients undergoing caesarean section for fetal compromise who developed spinal anaesthesia-induced hypotension were randomised to receive phenylephrine 100 µg or norepinephrine 8 µg for treatment of each hypotensive episode, defined as systolic blood pressure <100 mmHg. Umbilical cord arterial and venous blood samples were obtained for blood gas analysis. The primary outcome measure was umbilical artery pH. RESULTS: One hundred patients (50 in each group) were studied. There was no significant difference in umbilical artery pH between the two groups (mean difference 0.001; 95% CI -0.032 to 0.034). The number of hypotensive episodes, vasopressor boluses required, the incidence of bradycardia, heart rate and blood pressure trends following vasopressor administration, and the incidence of nausea/vomiting were not significantly different between groups. CONCLUSION: Phenylephrine 100 µg and norepinephrine 8 µg were not significantly different in terms of neonatal outcome when administered as intravenous boluses for treatment of spinal anaesthesia-induced hypotension in parturients undergoing emergency caesarean sections for fetal compromise.

Primary cutaneous large B-cell lymphoma shows activation of nuclear factor kappa B and low incidence of Epstein-Barr virus.

Primary cutaneous lymphomas are defined as lymphomas, which are present in the skin without evidence of extracutaneous disease at the time of diagnosis. Primary cutaneous large B-cell lymphoma (PCLBCL) is a subtype of primary cutaneous B-cell lymphoma with a female predominance, occurring in elderly patients and known to have unfavorable prognosis. We evaluated 10 cases of PCLBCL in immunocompetent patients between 2005 and 2008. A panel of immunoperoxidase stains; CD3, CD10, CD20, BCL2, BCL6, and MUM1 were performed on all cases. Nuclear factor kappa B (NF-kappaB) pathway activation was evaluated using an immunostain for P65. The presence of Epstein-Barr virus (EBV) was assessed using Epstein-Barr virus encoded RNA (EBER) in situ hybridization probe. All cases were CD20 positive and CD3 negative. CD10, BCL6, BCL2, and MUM1 were positive in 4/10 (40%), 6/10 (60%), 7/10 (70%), and 7/10 (70%) cases, respectively. NF-kappaB activation was detected in 7/10 (70%) cases. One (10%) case was positive for EBV by in situ hybridization. Interestingly, the EBV positive case was also positive for MUM1 and negative for CD10, indicating an activated immunophenotype. In conclusion, majority of PCLBCL shows activation of NF-kappaB pathway with a low incidence of EBV.

Comparison of the potency of phenylephrine and norepinephrine bolus doses used to treat post-spinal hypotension during elective caesarean section.

BACKGROUND: Phenylephrine, although considered the vasopressor of choice, can cause reflex bradycardia and a fall in cardiac output. Norepinephrine, due to its direct positive chronotropic and reflex negative chronotropic actions, is expected to overcome this problem. However, limited information about its effective dose for management of post-spinal hypotension, and its potency compared to phenylephrine, is available. METHODS: One hundred consecutive patients who developed post-spinal hypotension were treated with a predetermined dose of either phenylephrine or norepinephrine. Correction of hypotension after one minute was considered 'success'. The starting dose for the first patient and testing interval (the incremental or decremental dosing) were 100 µg and 10 µg in the phenylephrine group, and 6 µg and 0.5 µg in the norepinephrine group. Doses for subsequent patients were determined by the responses of previous patients according to the Narayana rule for up-down sequential allocation. ED95 and ED50 of phenylephrine and norepinephrine boluses and their potency ratio were calculated. RESULTS: Using Probit analysis, ED95 and ED50 values were 43.1 µg (95% CI 39.5 to 65.0 µg) and 33.2 µg (95% CI 5.1 to 37.0 µg) for phenylephrine, and 3.7 µg (95% CI 3.5 to 4.7 µg) and 3.2 µg (95% CI 1.8 to 3.4 µg) for norepinephrine. The relative potency ratio of norepinephrine and phenylephrine was 11.3 (95% CI 8.1 to 16.9). CONCLUSION: Based on the results of this study, norepinephrine is about 11 times more potent than phenylephrine. When used as bolus doses for treatment of hypotension, 100 µg phenylephrine should be approximately equivalent to 9 µg norepinephrine.

The mechanism of cyclosporine's action in the inhibition of testosterone biosynthesis by rat Leydig cells in vitro.

We have previously demonstrated that cyclosporine inhibits testosterone (T) biosynthesis in vivo. To better understand the mechanism by which CsA inhibits T synthesis, interstitial cells were isolated from rat testes and incubated in the standard medium 199 with or without CsA (0-10 micrograms/ml) in the presence or absence of human chorionic gonadotropin (hCG, 10(-7) M) and 8-bromo cyclic AMP (cAMP, 0.5 mM) for 3 hr at 32 degrees C. The levels of cAMP and T were determined by RIA. CsA did not inhibit the basal secretion of T, but inhibited hCG-stimulated T production in a dose-dependent manner (4 ng/10(6) cells vs. 10 ng/10(6) cells at a CsA dose of 5 micrograms/ml, P less than 0.05). Radioligand binding of 125I-hLH to testicular membranes was not affected by CsA, as CsA did not compete with hCG/LH for binding sites (25-28% binding with or without CsA). Similarly, the MIX-stimulated cAMP production was not affected by CsA (24.03 +/- 1.09 vs. 20.60 +/- 0.38 pmol/10(6) cells), suggesting that CsA does not inhibit the accumulation of the second messenger. However, when interstitial cells were incubated with CsA in the presence of cAMP, a significant dose-dependent decline in T secretion was observed (7 ng/10(6) cells vs. 20 ng/10(6) cells at a CsA dose of 5 micrograms/ml). To determine whether CsA inhibits the steps beyond cAMP stimulation of T secretion, the kinetic parameters (Km and Vmax) of steroidogenic enzymes, delta 4-3 keto-17 alpha hydroxylase (17 alpha-hydroxylase), and delta 4-3 keto-17 beta hydroxy steroid dehydrogenase (17B-HSD) were determined by using Michaelis Menten analysis. Results are shown in the presence of CsA vs. no CsA: Km and Vmax values for 17 alpha-hydroxylase were (2.32 vs. 7.98 microM) and (27.96 vs. 100.97 pmol/mg protein/min), respectively. For 17B-HSD the Km and Vmax were (2.14 vs. 1.52 microM) and (15 vs. 15 pmol/mg protein/min), respectively. These results indicate that CsA inhibits the activity of 17 alpha-hydroxylase uncompetitively and 17B-HSD activity competitively. In conclusion the primary site for CsA inhibition is the cAMP stimulation and, CsA inhibits T synthesis at multiple sites.

Comparison of secretion of catecholamines from the rat adrenal medulla during continuous exposure to nicotine, muscarine or excess K.

The effects of continuous exposure of chromaffin cells to nicotine, muscarine and excess potassium on the secretion of catecholamines and uptake of radiolabeled calcium were investigated in the isolated perfused adrenal gland of the rat. Perfusion with 20 microM nicotine-Krebs solution increased catecholamine secretion from a basal value of 5 ng to 273 ng in the first 5 min. The secretion declined to about 20 ng after 30 min of continuous exposure to nicotine (slow desensitization). 200 microM muscarine increased the secretion to about 150 ng within the first 5-10 min, and remained at the level of 110 ng even after continuous perfusion with muscarine for up to 40 min (no desensitization). About 700 ng of catecholamines were secreted within the first 5 min of exposure to 55 mM K, the secretion declined to about 350 ng in the next 5 min, and remained almost at this level for 120 min during continuous perfusion with excess K (partial desensitization). When the secretion of catecholamines was depressed by continuous exposure to nicotine for 30 min, introduction of 55 mM K or 200 microM muscarine (along with nicotine) still led to a large increase in the secretion of catecholamines. Omission of calcium ions from the perfusion medium containing either nicotine or 55 mM K caused a rapid decline in the secretion of catecholamines, suggesting influx of external calcium, but muscarine-induced secretion was only modestly reduced by calcium removal, suggesting mobilization of internal calcium. Addition of EGTA to calcium-free medium completely abolished the secretion evoked by all agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Demonstration of adrenergic and dopaminergic receptors in cultured sympathetic neurons--their coupling to cAMP but not to the transmitter release process.

Experiments were carried out on cultured sympathetic neurons of the chick embryo; first, to demonstrate the presence of adrenergic and dopaminergic receptors, and then to see if these receptors are involved in regulation of transmitter release. We show that alpha 2-agonists, norepinephrine, epinephrine and clonidine, had no effect on neuronal cyclic 3',5'-adenosine monophosphate content. Forskolin enhanced neuronal cyclic 3',5'-adenosine monophosphate from a control value of about 20 pmoles/mg protein to 150 pmoles/mg protein. In the presence of alpha 2-agonists and forskolin the cyclic 3,5'-adenosine monophosphate content increased between 340 and 430 pmoles/mg protein. The alpha 1-agonist, phenylephrine, had no such effect. The facilitatory effect of alpha 2-agonist on forskolin-stimulated cyclic 3',5'-adenosine monophosphate production was blocked by the alpha 2-antagonist, yohimbine, but not the alpha 1-agonist, prazosin. Dopamine did not affect neuronal cyclic 3',5'-adenosine monophosphate content, but forskolin-stimulated increase in cyclic 3',5'-adenosine monophosphate was further facilitated by dopamine, and this effect was blocked by haloperidol. Activation of neuronal alpha 2-receptors by norepinephrine, epinephrine and clonidine did not interfere with electrically induced release of tritium from [3H]-norepinephrine-loaded sympathetic neurons. However, if sympathetic neurons were co-cultured with heart cells, clonidine, norepinephrine and epinephrine markedly inhibited the stimulation-induced release. Yohimbine or phentolamine partially reversed the inhibitory effects of alpha 2-agonists. alpha 2-Agonists and -antagonists also modified stimulation-induced release of tritium from [3H]norepinephrine-loaded hearts of the chick embryo.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous secretion of catecholamines from the adrenal medulla and of [3H]norepinephrine from sympathetic nerves from a single test preparation: different effects of agents on the secretion.

The uptake and release of catecholamines was investigated in the isolated perfused adrenal gland of the rat after preloading the preparation with [3H]norepinephrine, and the effects of various agents were examined on the stimulation-evoked secretion of catecholamines and total tritium. Large quantities of tritium were found in the adrenal medulla after either intravenous injection of [3H]norepinephrine to the rat, or perfusion of the isolated adrenal gland with Krebs-bicarbonate solution containing [3H]norepinephrine. The retention of the tritium was inhibited 90% by desipramine. Acute treatment with guanethidine and chronic treatment with 6-hydroxydopamine abolished the secretion of tritium without affecting the secretion of catecholamines evoked at 1 Hz. Nicotine, muscarine and acetylcholine enhanced the secretion of catecholamines but not tritium, whereas tyramine and ephedrine enhanced the secretion of tritium but not catecholamines. It is concluded that chromaffin cells do not possess the norepinephrine uptake mechanism and that the uptake of [3H]norepinephrine occurs mainly in sympathetic nerve terminals present in the adrenal gland and the surrounding blood vessels (adrenal and renal veins). The differential localization of [3H]norepinephrine and catecholamines allowed us to test the effects of a variety of pharmacological agents that alter neurotransmitter release by acting on receptors on the neuronal membrane, acting on sodium and potassium channels, or acting to alter the intracellular concentrations of adenosine 3',5'-cyclic monophosphate and protein kinase C. Transmural stimulation (1 Hz for a total of 300 pulses) markedly enhanced the release of catecholamines and tritium which was blocked by tetrodotoxin (sodium channel-blocker) and potentiated by tetraethylammonium and gallamine (potassium channel-blockers). Phentolamine, an alpha adrenergic blocking agent which acts on both alpha-1 and alpha-2 receptors, caused a 3- to 4-fold facilitation of the tritium secretion while inhibiting catecholamine secretion by 45%. [Met]enkephalin almost completely inhibited the evoked-secretion of tritium but had very little effect on the secretion of catecholamines. Forskolin inhibited the tritium secretion by 80% but produced more than a 2-fold facilitation of catecholamine secretion. Phorbol 12,13-dibutyrate caused facilitation of evoked secretion of both catecholamines and tritium. A combination of phorbol ester and forskolin had a synergistic effect on stimulation-evoked secretion of catecholamines, whereas phorbol ester partially reversed the inhibitory effects of forskolin on the tritium secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Antiproliferative effects of c-myc antisense oligonucleotide in prostate cancer cells: a novel therapy in prostate cancer.

OBJECTIVES: To explore the possibility of using antisense oligonucleotide therapy for prostate cancer, we investigated the effect of c-myc-antisense-oligonucleotide (c-myc-As-ODN) in human prostate cancer cell lines such as LNCaP, PC3, and DU145. METHODS: LNCaP, PC3, and DU145 cells were incubated in the presence of c-myc-As-ODN. Dose (0 to 10 microM) and time dependent (1 to 6 days) effects on proliferation and viability were examined by [3H]thymidine incorporation and MTT assay, respectively. Flow cytometry analysis was carried out to analyze cell cycle status by determining the DNA content in LNCaP cells. Control cultures received either c-myc-sense-ODN or scrambled (nonsense) nucleotides. RESULTS: Time- and dose-dependent decreases in DNA synthesis and cell viability were noted for all three prostate cancer cell lines after c-myc-As-ODN treatment. Further studies using LNCaP cells indicated that these changes were accompanied by an increase in the percentage of cells with less than 2N DNA content after c-myc-As-ODN treatment. The results suggest that c-myc-As-ODN induces cell death. Comparison of a c-myc-As-ODN-treated group with a group subjected to isoleucine deprivation revealed that thymidine incorporation was almost the same in c-myc-As-ODN-treated LNCaP cells and in LNCaP cells at early S phase. CONCLUSIONS: These results suggest that c-myc-As-ODN inhibits prostate cancer cell growth and proliferation mainly by decreasing cell viability.

Secretion of catecholamines from the perfused adrenal gland of the rat is not regulated by alpha-adrenoceptors.

The question of local regulation of catecholamine secretion by alpha-adrenoceptors of either presynaptic splanchnic nerve terminals or postsynaptic chromaffin cells was investigated in the isolated perfused adrenal gland of the rat. The most selective agonists and antagonists of alpha-adrenoceptors such as clonidine, phenylephrine, yohimbine, etc. did not modify the secretion of catecholamines evoked by splanchnic nerve stimulation or nicotine. It is concluded that the negative feedback mechanism that controls the release of norepinephrine at the central and peripheral noradrenergic synapses does not operate in the adrenal medulla to locally control the secretion of medullary hormones.

Veratrine supports the in vitro survival of embryonic chick sympathetic neurons.

Veratrine (VT), an alkaloid known to act on the sodium channels and cause depolarization of a cell membrane, was found to support the survival of cultured sympathetic neurons. At 30 microM it was as effective as nerve growth factor (NGF), as determined by the cell counts and [3H]norepinephrine ([3H]NE) uptake. Protein kinase C (PKC) activity of the surviving neurons was measured because of our previous finding that depolarizing concentrations of K+ support the survival and cause several fold increase in the enzyme activity. An acute treatment of NGF-supported sympathetic neurons by VT did not alter PKC activity.

Effects of subchronic treatment with cis-platinum on testicular function, fertility, pregnancy outcome, and progeny.

Cis-platinum-based chemotherapy is known to impair spermatogenesis, but the effects of paternal cis-platinum treatment on the progeny are unknown. To study this effect, sexually mature male Sprague-Dawley rats were administered intraperitoneal injections of saline or cis-platinum (0.5 mg/kg per day) for 9 weeks. Every week, one set of control and treated animals was mated with females in proestrus. Nineteen days later, the females were subjected to laparotomy, and the numbers of corpora lutea, resorptions, and normal and abnormal fetuses were noted. In conjunction, the effects of treatment on the hypothalamo-pituitary-gonadal axis of the treated males were evaluated. Cis-platinum-treated animals failed to grow; the weights of the reproductive organs and the sperm counts declined from week 2 onward, and sperm motility was reduced throughout the testing period. Circulating and intratesticular levels of testosterone declined from week 3 of treatment and follicle-stimulating hormone levels were not affected. Serum levels of luteinizing hormone declined from week 3 and were not detectable from week 6 onward. However, the pituitary response to gonadotropin-releasing hormone was intact in all treated groups. There was no significant decrease in fertility, but a prominent increase in pre- and postimplantation losses of fetuses after cis-platinum treatment was observed. There was also a decrease in the male-to-female ratio of the offspring. A small but significant number of malformed and growth-retarded fetuses was also found among the offspring of cis-platinum-treated males. These results suggest that subchronic treatment with low doses of cis-platinum may affect progeny; such effects are seen in addition to the apparent alteration in a number of measures of reproductive function of treated males.

Phorbol ester, an activator of protein kinase C, enhances calcium-dependent release of sympathetic neurotransmitter.

The effect of phorbol ester, phorbol 12,13-dibutyrate, was investigated on the overflow of tritium from 3H-noradrenaline-loaded sympathetic neurons of the isolated perfused salivary gland of the rat. Stimulation (1 Hz for 60 s)-evoked overflow of tritium was enhanced by phorbol ester. A significant enhancement was seen at 1 nmol/l, which increased to a maximum level (over 4-fold) at 30 nmol/l. The spontaneous overflow of radioactivity, however, was not affected by any concentration of phorbol ester. The facilitatory effect of phorbol ester on stimulation-evoked overflow was observed in the presence of inhibitors of neuronal and extraneuronal uptake as well as after removal of negative feedback inhibition of release by presynaptic alpha-adrenoceptors. Tyramine (7 mumol/l for 10 min) caused a marked increase in the overflow of tritium in either the presence or absence of calcium. However, tyramine-induced overflow was not enhanced by phorbol ester. It is concluded that protein kinase C of sympathetic neurons is involved in an exocytotic release of the transmitter.

Facilitation of noradrenaline release by gallamine in the rat salivary gland.

The effect of gallamine on spontaneous and stimulation-evoked overflow of tritium was studied in the submandibular gland of the rat. The gland was perfused retrogradely and labeled with 3H-noradrenaline. The stimulation-evoked (1 Hz for 60 s) overflow of tritium was facilitated by increasing concentrations of gallamine (0.3-20 mM). None of the concentrations of gallamine increased the spontaneous overflow of the tritium. The facilitatory effect of gallamine was observed in 0.3 to 5 mM calcium medium; the maximum facilitation was observed at the normal concentration of calcium (2.5 mM). The facilitatory effect of gallamine was inversely related to the frequency of stimulation (10-fold facilitation at 1 Hz and 3-fold at 10 Hz). Stimulation of the salivary gland by a single pulse (1 ms duration) in the normal medium did not evoke an overflow of tritium; however, the same stimulus produced a marked increase in the overflow in the presence of gallamine. The facilitatory action of gallamine on the release of sympathetic transmitter is ascribed to the enhanced availability of calcium ions to the secretory process resulting from blockade of potassium conductance during nerve activity.