Assessing the impact of evolving evidence in renal cell carcinoma treatment: an update of the Renal Cell Carcinoma Appropriateness-based Treatment Toolkit (ReCATT).

The appropriateness of the numerous therapeutic options available for patients with advanced or metastatic renal cell carcinoma (RCC) was evaluated in 2011, using the RAND/University of California, Los Angeles (UCLA) appropriateness methodology to match treatment suitability to a range of patient scenarios. However, the RCC therapeutic area evolves rapidly and a body of new clinical data has accrued in the intervening years; as a result the exercise was repeated in 2013 using the same methodology, expert panel and patient scenarios. The aim of the updated assessment was to update the guidance to clinicians and use it to develop an interactive web-based application, the Renal Cell Carcinoma Appropriateness-based Treatment Toolkit (ReCATT). This round of assessment achieved greater concordance concerning the appropriateness of treatments/interventions for the clinical scenarios tested; this higher level of agreement is likely to reflect the body of scientific evidence accrued since the previous assessment exercise. Many of the areas of disagreement in 2011 related to the suitability of pazopanib or sunitinib treatment; in the 2013 assessment both agents were considered appropriate treatment options for many of the clinical scenarios assessed. Uncertain scenarios often are related to the optimal management of metastatic RCC with clear cell histology. The use of the RAND/UCLA RCC assessment findings to develop the ReCATT support tool will help to disseminate expert opinion concerning best treatment practice and guide the clinical management of RCC patients treated in the community setting.

Evaluation of treatment options for patients with advanced renal cell carcinoma: assessment of appropriateness, using the validated semi-quantitative RAND corporation/University of California, Los Angeles methodology.

A diverse range of treatment options and interventions are available for the management of renal cell carcinoma (RCC), allowing clinicians to tailor therapy to best meet their patient's needs and situation. However, choosing from the plethora of options can be problematic. RCC treatment guidelines advise on the most efficacious agents based upon specific clinical trial populations, but these do not always take into account all the patient factors that influence the suitability of treatment options for individual patients. This study used the validated RAND/UCLA (RAND corporation/University of California, Los Angeles) 'appropriateness methodology' to integrate clinical efficacy data with expert opinion concerning the use of specific RCC treatment options for particular patient scenarios, in an attempt to facilitate the widespread implementation of patient-focussed treatment choices. Use of the methodology has allowed us to develop treatment algorithms for patients with locally-advanced RCC and for those with metastatic disease post-nephrectomy or with primary tumour in situ. The algorithms take into account patient-specific characteristics such as tumour histology, prior treatment and known risk factors to advise whether a particular treatment intervention is appropriate, not appropriate or of uncertain appropriateness. Use of this methodology aims to develop a formalised process by which expert opinion can be integrated with clinical data and used as an additional source of information that can provide further guidance concerning difficult treatment decisions when data are absent or sparse.

Ion dependence of the release of noradrenaline by tetraethylammonium and 4-aminopyridine from cat splenic slices.

Cat splenic slices prelabelled with [3H]-noradrenaline were incubated in oxygenated Krebs-bicarbonate solution at 37 degrees C, and the spontaneous total 3H release into different incubation media monitored. In normal Krebs bicarbonate solution, the spontaneous tritium fractional release amounted to 3.7% of the tissue radioactivity content per 5 min collection period. Tetraethylammonium (TEA) increased spontaneous transmitter release in a concentration-dependent manner; the release was maximal at 30 mM and was 3.5 times the basal release. 4-Aminopyridine (4-AP) also enhanced the spontaneous release of tritium. The response increased linearly with 4-AP concentration (1-10 mM). With 10 mM 4-AP, the release was as much as 6 times the basal transmitter release. Guanidine was much less potent than either TEA or 4-AP. The secretory response to TEA or 4-AP was little affected by changes in external Ca2+, Mg2+, Na+, Cl-, H2PO4- or by tetrodotoxin. However, transmitter release evoked by TEA or 4-AP strongly depended upon the concentration of HCO3- of the incubation solution; in fact, the secretory response varied almost linearly between 1 and 25 mM HCO3-. The mechanisms underlying these effects are probably related to the well-known ability of TEA and 4-AP to block K+ conductance that would cause depolarization of the splenic sympathetic nerve terminals. The HCO3- requirements for the secretory response are probably related to the ability of CO2/HCO3- solutions to mobilize and release Ca2+ from intracellular organelles.

The release of acetylcholine and of catecholamine from the cat's adrenal gland.

The cat's adrenal gland was perfused in situ with Krebs solution containing eserine; the amount of acetylcholine and of catecholamine released was measured. Splanchnic nerve stimulation (5 Hz for 2 min) increased the release of acetylcholine and catecholamine; the molar ratio of evoked release of catecholamine to acetylcholine was 122 +/- 8. It is suggested that this amplification is achieved because a chromaffin cell granule contains more mediator than does the acetylcholine quantum that releases it. The release per impulse of catecholamine during splanchnic nerve stimulation at 30 Hz was less than that released by stimulation at 1 or 5 Hz. This depression is attributed to a presynaptic failure, because the release of acetylcholine was similarly frequency dependent. The release of catecholamine was linearly related to the release of acetylcholine over the range tested, indicating that the input-output relationship at the splanchnic-adrenal medullary junction is linear. During continuous stimulation of the splanchnic nerve (5 Hz), catecholamine release declined to a level that was 32 +/- 2% of the initial output. This fatigue is attributed primarily to a postsynaptic depression, because the release of acetylcholine was maintained at 71 +/- 6% of its initial level. The presence of eserine in the perfusate was necessary for the release of acetylcholine to be detected, but in the presence of eserine catecholamine release was 90 +/- 10% that in the drug's absence. It is concluded that released acetylcholine is hydrolysed at some distance from its site of release and action. Glands perfused with raised K+ released acetylcholine and catecholamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Orthograde and retrograde axonal transport of calmodulin in a cat noradrenergic neurone.

Subcellular distribution studies of calmodulin in cat sympathetic ganglia demonstrated that about 90% of the protein remained in the 27,000 g supernatant, suggesting that it is a cytosolic protein. Only 4.5% was recovered in the microsomal fraction pellet. The inferior mesenteric ganglia contained 93.3 +/- 3 ng calmodulin per ganglion, and segments of unligated cat hypogastric nerves had 6.53 +/- 0.32 ng per 5 mm segment. When the nerve was ligated in the middle and left in the cat for 1-6 days, substantial amounts of calmodulin accumulated in segments of nerve immediately proximal (P1) and distal (D1) to the ligature. The amounts found in P1 amounted to 15.3, 20, 30.4 and 39.4 ng calmodulin per 5 mm segment 1, 2, 3 and 6 days after ligation, respectively. The average rate of transport was 5.5 mm per day, which corresponds to a slow component b of axonal transport (SCb). The accumulation of calmodulin in D1 was also increased with the time of ligation. After 1, 2, 3 and 6 days, the amounts of the protein found in D1 were 14.4, 17.7, 19 and 21 ng per 5 mm segment, respectively. The calculated mean rate for the retrograde transport was 3.9 mm per day. Decentralization of the inferior mesenteric ganglia did not affect the rate of accumulation of calmodulin or the basal amounts found in ganglia and nerves. Local injection inhibited the orthograde, but not the retrograde axonal transport of the protein. It is concluded that calmodulin undergoes a process of slow orthograde axonal transport probably incorporated into the axoplasmic matrix of a network of actin microfilaments. The protein is also transported in a retrograde manner.

Support for a role for feedback regulation of norepinephrine release.

There is abundant evidence that norepinephrine (NE) and other sympathomimetic amines with alpha-adrenoceptor activity inhibit the electrically evoked release of NE, whereas phenoxybenzamine and other alpha-adrenergic blocking agents enhance the electrically evoked release of NE. The physiological relevance of these observations, however, is disputed. The intent of this paper is to show that alpha-adrenergic blocking agents generally enhance transmitter output on nerve stimulation, but that some are more selective for presynaptic than for postsynaptic alpha receptors. Suggestions are made to account for the modulation of NE release as evoked by a single pulse.

Pharmacological dissection of receptor-associated and voltage-sensitive ionic channels involved in catecholamine release.

The experiments were designed to quantify pharmacologically the degree of participation of channels associated with the nicotinic cholinoceptor compared with voltage-sensitive channels during the evoked release of [3H]noradrenaline from prelabelled 3-7-day old cultured bovine adrenal chromaffin cells. To achieve this purpose we studied (a) the release of [3H]noradrenaline evoked by secretagogues known to trigger the secretory response through activation of receptor-associated channels (acetylcholine, nicotine), voltage-sensitive Na+ (veratridine) and Ca2+ (high [K+] ) channels or direct, channel-independent promotion of Ca2+ entry (ionomycin); and (b) the selective blockade of some of those responses using ionic manipulations (Na+ deprivation, high Mg2+) or drugs known to block the activity of receptor-operated channels (imipramine, cocaine), voltage-dependent Na+ (tetrodotoxin) or Ca2+ (nitrendipine) channels. Inhibition by nitrendipine, a potent Ca2+ antagonist, of the secretory responses to both nicotine and high [K+] indicates a preferential Ca2+ entry through voltage-sensitive channels during the secretory process. Blockade by cocaine and imipramine of the release of [3H]noradrenaline evoked by acetylcholine and nicotine, without alteration of the responses to high [K+], veratridine or ionomycin, speaks in favor of a selective inactivation of the nicotinic receptor-associated channel. Since Na+ deprivation abolished [3H]noradrenaline release produced by nicotine, it seems that Na+ entry through the receptor-linked ionophore might be a primary event in the initiation of the secretory process; the fact that tetrodotoxin did not affect the release favors this view. However, veratridine induced a tetrodotoxin-sensitive secretory response, suggesting the presence of voltage-sensitive Na+ channels which might physiologically be used to propagate action potentials through gap junctions between adjacent chromaffin cells, only in the intact gland.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of 6-hydroxydopamine on bovine adrenal chromaffin cells in culture.

The effect of 6-hydroxydopamine on the catecholamine content and cell morphology of bovine adrenal chromaffin cells in culture was investigated. 6-Hydroxydopamine markedly reduced the catecholamine content of the cultured chromaffin cells after 6 and 24 h exposure. The effect was dose-related, with half-maximal depletion occurring at 2.4 X 10(-5) M. Cells exposed to 6-hydroxydopamine for 3 h and then to normal medium without the drug for 3 h more showed the same degree of toxicity as cells exposed to the drug for the entire 6 h. Ascorbate at high concentrations also exhibited toxicity toward chromaffin cells between 6 and 24 h of exposure. 6-Hydroxydopamine produced marked changes in cell morphology. At 1 h the cells appeared normal, at 3 h the processes were markedly shortened, and at 6 h they were completely retracted. On exposure for 24 h there were gross morphological changes and most cells were detached and free-floating in the medium. The toxicity of 6-hydroxydopamine in bovine adrenal chromaffin cells is discussed.

Modification of potassium-evoked release of noradrenaline by various ions and agents.

1 Release of noradrenaline (NA) from isolated spleen slices of the cat by high K(+) and tetraethylammonium (TEA) was investigated. Studies were conducted with spleen slices whose tissue stores were prelabelled with [(3)H]-noradrenaline ([(3)H]-NA).2 Release by high K(+) was related to the K(+) concentration of the incubation medium. Release of [(3)H]-NA by 28.5 mM K(+) was only barely detectable over the background, while 70 mM K(+) enhanced release to more than 600% of the background output. Tetrodotoxin (TTX) did not block responses to 28.5 or 35 mM K(+).3 Background release was not modified by 1 or 3 mM TEA, but 10 and 30 mM TEA enhanced the release of [(3)H]-NA by about 50% and 150%, respectively, over the background level. Neither TTX nor hexamethonium (C(6)) blocked the TEA response. Release by TEA was also not blocked in Ca(2+)-free medium or in Ca(2+)-free medium containing up to 3 mM EGTA. Release by TEA was blocked in Ca(2+)-free medium containing 5 mM EGTA, and by La(3+) or Mn(2+).4 The response to 35 mM K(+) was not modified by 1 or 3 mM TEA; 10 mM TEA had an additive effect; and 30 mM TEA with 35 mM K(+) produced a response which was greater than the simple sum of responses to 35 mM K(+) and 30 mM TEA. At 45 mM K(+), 3 and 10 mM TEA potentiated the response, and at 30 mM K(+) only 1 mM TEA showed potentiation. TTX did not alter the response to high K(+) plus TEA.5 When TEA (30 mM) was added during prolonged incubation with 140 mM K(+), the response was only slightly enhanced. This suggests that a large part of the secretory response to TEA is mediated through mobilization of Ca(2+) activated by depolarization.6 Phenoxybenzamine (3.3 muM) potentiated responses to 35 and 140 mM K(+) by about 50%, and TTX did not influence this potentiation. Acetylcholine (ACh) blocked responses to 28.5 and 35 mM K(+), and 1 mM TEA antagonized this ACh blockade.7 In the perfused adrenal gland of the cat, the secretory response to TEA was related to its concentration. The response was not diminished by low Na(+), TTX, or C(6), but was markedly attenuated when TEA was applied 10 min after the start of perfusion with high K(+).

Presence and axonal transport of cholinoceptor, but not adrenoceptor sites on a cat noradrenergic neurone.

1. Noradrenaline release and radioligand binding studies were carried out in the cat hypogastric nerve ligated in vito 2 cm distal to the inferior mesenteric ganglion for different time periods, and in different effector organs.2. Large quantities of noradrenaline and dopamine beta-hydroxylase (DBH) accumulated in the segments of nerve immediately proximal (P(1)) and distal (D(1)) to the ligation, with rates of about 100 and 25 mm/24 hr for the orthograde and retrograde transport, respectively.3. Nicotine evoked the release of noradrenaline from P(1) and atrial slices; the secretory response to nicotine was completely antagonized by mecamylamine. [(3)H]alpha-bungarotoxin biding to membranes from P(1) allowed the estimation of a K(D) of 2.97 nm and a B(max) of 1639 f-mole/mg protein.4. Acetylcholine inhibited the release of endogenous noradrenaline evoked by high K(+) stimulation in atrial slices, but not in P(1) segments. Similarly, carbachol decreased [(3)H]noradrenaline release induced by electrical stimulation (twenty-six shocks, 2 Hz, 5 msec) in the atrium but not in P(1).5. [(3)H]Quinuclydinilbenzylate ([(3)H]QNB) specifically binds to membranes from P(1) and vas deferens, following a saturation curve. In the case of P(1) segments taken 48 hr after ligation a K(D) of 0.35 nm and a B(max) of 129 f-mole/mg protein were found.6. The fact that the B(max) in P(1) and D(1) increased with the time of ligation suggests that orthograde and retrograde axonal transports of muscarinic binding sites exist in this nerve, with approximate rates of transport of 15 and 8 mm/24 hr, respectively.7. As far as adrenoceptors are concerned, we observed that yohimbine or phentholamine did not modify transmitter release from P(1), evoked by high K(+) or electrical stimulation. However, yohimbine enhanced the release of [(3)H]noradrenaline induced by electrical stimulation from splenic slices of the same animals.8. [(3)H]Clonidine, [(3)H]dihydroergocryptine or [(3)H]dihydroalprenolol ([(3)H]DHA) did not specifically bind to membranes from P(1), in spite of the fact that they showed typical saturation curves for specific binding in cortex and atrial membranes from the same cats.9. In conclusion, these data (a) further show that the ligated hypogastric nerve is a good model of noradrenergic nerve terminal free of effector cell; (b) provide direct evidence for the neural location of nicotinic receptors whose activation trigger noradrenaline release from noradrenergic neurones; (c) demonstrate the neural location and axonal transport of muscarinic receptor sites, but leave certain doubts about its functional role in this noradrenergic neurone; and (d) do not support the hypothesis that alpha and beta-adrenoceptors which modulate noradrenaline release from peripheral noradrenergic nerve terminals are neurally (or prejunctionally) located.

Effect of muscarine on release of catecholamines from the perfused adrenal gland of the cat.

1 The secretory effect of muscarine was studied in the perfused adrenal gland of the cat. During perfusion of the adrenal gland with Krebs-bicarbonate solution containing muscarine 480 microM, the rate of catecholamine (CA) secretion was 2.02 +/- 0.43 micrograms/2 min in the first 2 min; thereafter, CA output declined only moderately, to reach about 70% of the initial value after 10 min. Secretory responses to brief infusions of muscarine remained reproducible for at least the first 3 infusions. 2 When the adrenal gland was perfused with muscarine (480 microM), infusions of high K+, nicotine, or veratridine produced their usual responses. A 100 fold lower dose of muscarine also failed to modify these responses. 3 During perfusion with high K+, muscarine evoked a secretory response that was only slightly smaller than the response to muscarine alone. 4 It is concluded that muscarine and nicotine activate CA secretion in the cat adrenal gland by independent mechanisms and that the muscarinic response, unlike the nicotinic response, is not readily desensitized.

Ionomycin stimulates secretion of catecholamines from cat adrenal gland and spleen.

Ionomycin, a polyether antibiotic, stimulated the secretion of catecholamines and dopamine beta-hydroxylase from perfused adrenal glands and [3H]norepinephrine ([3H]NE) from spleens of the cat. Release was calcium dependent, and strontium or barium did not substitute for calcium. Ionomycin failed to release [3H]NE from reserpinized spleens. High magnesium did not interfere in the ionomycin response, but lanthanum and manganese blocked it. Ionomycin response that was pH dependent was not affected by potassium depolarization. The secretory response to ionomycin was enhanced when both glycolysis and oxidative metabolism were inhibited. It is concluded that ionomycin introduces calcium into the chromaffin cells and adrenergic nerve terminals to cause the secretory response and that a rise in intracellular calcium may be an adequate stimulus for secretion.

Adrenergic nerve-blocking activity of a new guanidine derivative.

The effects of 4-7-exo-methylene-hexahydroisoindoline-ethyl guanidine hemisulfate (no. 865-123) on norepinephrine release were investigated in the perfused spleen on the cat. No. 865-123 irreversibly blocked the release of norepinephrine evoked by nerve stimulation. Tetraethylammonium, 4-amino-pyridine and guanidine readily reversed this inhibitory effect, and the norepinephrine output was nearly tripled after repeated stimulation of the nerves. On subsequent perfusion with Krebs' solution without any drugs, the inhibitory effect of no. 865-123 partially reappeared. Perfusion pressure responses followed the same pattern as release except during the final perfusion period with Krebs' solution. It is suggested that tetraethylammonium, 4-aminopyridine and guanidine allow greater than normal amounts of calcium to accumulate inside the adrenergic nerve terminals during an action potential to reverse no. 865-123 blockade of norepinephrine release.

Potentiation of K+-evoked catecholamine release in the cat adrenal gland treated with ouabain.

1 A vigorous catecholamine secretory response was evoked by small increments (2-10 mM) of the extracellular concentration of K+ ([K+])o) in cat adrenal glands treated with ouabain (10(-4) M), and perfused with Krebs-bicarbonate solution at room temperature. 2 The secretory response depends on [K+]o; increments of [K+]o as small as 2 mM for 2 min evoked a clear secretory response; at 10-17.7 mM K+, the maximal secretory response was observed. In normal glands, not treated with ouabain, no increase of the rate of catecholamine output was observed by raising [K+]o up to 17.7 mM for 2 min. 3 The K+ secretory response was time-dependent, requiring at least 1 min to be initiated; on continued exposure to 10 mM [K+]o, the enhanced response remained for at least 1 h. 4 In low [Na+]o, the K+-secretory response was unchanged. However, in 0-Ca2+, high-Mg2+ solutions, or in the presence of D600, an organic Ca2+ antagonist, it was abolished. 5 The K+-induced secretory response was not altered in the presence of tetrodoxin or tetraethylammonium. 6 It is concluded that ouabain potentiated the catecholamine secretory response to raised [K+]o by increasing the amount of Ca2+ available to the secretory machinery through (a) mobilization of an enhanced pool of membrane-bound Ca2+, (b) activation of membrane Ca2+ inward current; or (c) decrease of intracellular Ca2+ buffering systems. The activation by ouabain of a membrane Na+-Ca2+ exchange system is not involved in this K+-secretory response. It is suggested that the plasma membrane ATPase enzyme system, by changing the affinity of its Ca2+ binding sites, might control the availability of this cation to the secretory machinery and, therefore, modulate catecholamine secretion in the adrenal gland.

Release of endogenous noradrenaline from ligated cat hypogastric nerve by veratridine.

The effect of veratridine on in vitro release of noradrenaline (NA) from ligated cat hypogastric nerve was investigated. After in vivo ligation for 24-48 h, large amounts of NA and dopamine-beta-hydroxylase (DBH) accumulated in the nerve segment immediately proximal to the ligature (P1). In vitro incubation of ligated nerves (segments P1 and P2) in oxygenated Krebs solution at 37 degrees C in the presence of veratridine caused a marked and dose-dependent release of endogenously accumulated NA into the incubation medium. The release continued to occur for a considerable time, even after washout of the drug. Veratridine-induced depletion of tissue NA was accounted for by its release, as NA, into the incubating medium. The secretory response to veratridine was readily blocked by tetrodotoxin (TTX). Veratridine-induced release was dependent on calcium and abolished by high magnesium. On the basis of the similarity between the NA secretory response to veratridine in this preparation and in adrenergically innervated organs, the results favour the view that the in vivo-ligated cat hypogastric nerve may serve as a useful model of adrenergic nerve terminals free of effector cells.

Action of nicotine on sympathetic nerve terminals.

Interaction between 4-aminophrydine (4-AP) and nicotine on sympathetic nerve terminals was studied in the isolated cat spleen slices, labeled with [3H]norepinephrine ([3H]NE). Incubation of slices for 5 min at 37 degrees C in low (50 microM) and high (2 mM) concentrations of nicotine released 0.8 +/- 0.08 and 2.73 +/- 0.39% of tissue [3H]NE. Tetrodotoxin (TTX) blocked the response to low nicotine but not to high nicotine. Low nicotine did not release [3H]NE in the absence of calcium. Response to high nicotine which persisted in calcium-free solution was blocked by ethylene glycol bis(beta-aminoethyl ether)N,N'-tetra-acetic acid. 4-AP (1 mM) not only enhanced the response to low nicotine but it effectively antagonized the suppressant effects of TTX and calcium-free solution on release induced by low nitotine. Restoration of release by 4-AP from TTX-blocked preparations occurred in the absence of calcium in the perfusion medium, but lanthanum (1 mM) blocked it. Restoration of release from spleen slices incubated in calcium-free Krebs' solution by 4-AP was blocked by lanthanum and prolonged incubation in calcium-free ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraaectic acid solution. It is concluded that at lower doses nicotine, by acting on nicotinic receptors, depolarizes the sympathetic nerve terminals to set off propagated action potentials which are responsible for NE release, and that 4-AP restores nicotine response in the presence of TTX or in the absence of calcium by mobilizing calcium both from extracellular and intracellular sources.

Release of noradrenaline from the ligated cat hypogastric nerve.

After in vivo ligation for 24 of the cat hypogastric nerve, large amounts of noradrenaline (NA) and dopamine beta-hydroxylase (DBH) accumulated in the nerve segment immediately proximal to the ligature (P 1). In vitro incubation of 24-h-ligated nerves ((segments P1 and P2) in oxygenated Krebs solution at 37 degrees C in the presence of the ionophore X537A or high K+ concentrations caused a marked release of endogenously accumulated NA into the incubation medium. High-K+-evoked release was entirely dependent on extracellular Ca2+. Electrical nerve stimulation caused an 80% tissue NA loss, but the transmitter could not be found in the medium as intact NA. These results suggest that the in vivo ligated cat hypogastric nerve may serve as a useful model of adrenergic nerve terminals free of effector cells.