Ability of nondepolarizing neuromuscular blocking drugs to act as partial agonists at fetal and adult mouse muscle nicotinic receptors.

We studied the ability of four nondepolarizing neuromuscular blocking agents (atracurium, gallamine, metocurine, and pancuronium) to act as competitive antagonists at mouse adult- and fetal-type muscle nictinic receptors. Receptor subunits for the fetal type (alpha, beta, gamma, and delta) and adult-type (alpha, beta, epsilon, and delta) receptors were stably expressed in quail fibroblasts. Binding for each drug was determined by the ability of the agents to reduce the initial rate of labeled alpha-bungarotoxin binding, and functional consequences were determined with the use of voltage-clamp studies of their ability to elicit currents or to block currents elicited by acetylcholine. Each agent has a different affinity for the two acetylcholine-binding sites on a single receptor; the rank order of affinities is the same for both fetal- and adult-type receptors. All agents inhibited activation of adult-type receptors by ACh, consistent with the idea that occupation of either the high or low affinity site completely blocks activation when acetylcholine binds to the other site on the receptor. The concentration dependence of the inhibition of acetylcholine-elicited current was predictable from the affinities estimated from independent measurements of the inhibition of alpha-bungarotoxin binding. Gallamine and pancuronium also acted as competitive inhibitors of fetal-type receptors, and, again, the concentration dependence of the inhibition was predictable from binding data. However, metocurine and atracurium could potentiate the responses of fetal-type receptors to low concentrations of acetylcholine. The interaction of metocurine and atracurium with acetylcholine at fetal-type receptors could be accounted for by a weak partial agonist activity. It has been suggested that some pairs of nondepolarizing neuromuscular blocking agents might be more efficacious because the high affinity site for one agent might be the low affinity site for another. This hypothesis was tested for the pair of agents metocurine and gallamine by determining the ability of a mixture of agents to inhibit the binding of alpha-bungarotoxin. The results are consistent with the idea that both metocurine and gallamine have a high affinity for the same site on the receptor. The ability of gallamine to block the partial agonist action of metocurine at fetal-type receptors was tested as well and also indicated that both agents share the same high affinity site.

Selection of stably transfected cells expressing a high level of fetal muscle nicotinic receptors.

We had earlier found that the numbers of mouse muscle nicotinic receptors expressed on the surface of individual cells of a stably transfected clonal line of quail fibroblasts varied from cell to cell (Kopta and Steinbach: J Neurosci 14:3922-3933, 1994). We have now used repeated selective passages of these clonal cells to produce a population of cells which expresses a greater and more uniform number of surface receptors per cell. The increased level is stable over many cell divisions, and over many half-lives for the metabolic degradation of the surface receptors. Selection was performed by adhesion to a surface coated with a monoclonal antibody to a surface epitope on the muscle receptor, followed by expansion of the most tightly attached population of cells. Studies of the selected cells show that the surface receptors contain all four subunits of the muscle nicotinic receptor, and the functional properties of the receptors appear normal. The metabolic stability of the surface receptors is not altered, while the amount of mRNA for the subunits is increased in the selected population of cells. These observations indicate that the more likely reason for increased expression is a transcriptional effect, and that translational or posttranslational changes are unlikely.

The conductance of the muscle nicotinic receptor channel changes rapidly upon gating.

We have recorded single-channel currents through fetal-type muscle nicotinic receptor channels at recording bandwidths of approximately 50 and 75 kHz. The time course of the rising phase of aligned and averaged openings can be entirely accounted for if it is assumed that the conductance of the single channel changes instantaneously, and that alignment and averaging introduce a dispersion of 2-3 microseconds. We conclude that we find no evidence for a gradual change in conductance as a channel opens or closes. The shapes of averaged power spectra are consistent with this conclusion, insofar as they exclude an exponential relaxation in the transition with a time constant of 10 microseconds or more.

The actions of the kappa 1 opioid agonist U-50,488 on presynaptic nerve terminals of the chick ciliary ganglion.

The actions of the kappa 1 opioid receptor agonist U-50,488 (trans-(+-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benz ene - acetamide methane sulfonate) on the membrane properties of presynaptic calyciform nerve terminals of the chick ciliary ganglion were examined using intracellular recordings obtained from intact ganglion preparations maintained in vitro. U-50,488 produced a concentration-dependent (30-1000 microM) hyperpolarization with an apparent increase in input resistance. This hyperpolarization resulted from inhibition of the Na(+)-K+ inward rectifier, since it was blocked by 3 mM Cs+ and was not observed when terminals were depolarized beyond resting potential where inward rectification was voltage inactivated. A depolarizing effect on membrane potential with a further rise in input resistance was commonly observed at the highest perfused U-50,488 concentration (1 mM). The depolarizing event appears to result from a decrease in membrane potassium conductance, as the reversal potential for the response was estimated to be between -70 and -90 mV and the potassium channel blocker Ba2+ (1 mM) abolished the response. The kappa 1 opioid receptor agonist also blocked spontaneously occurring miniature hyperpolarizations in the terminals, which are considered to be due to a Ca(2+)-dependent K+ conductance. Most of the responses to U-50,488 were abolished in the presence of the kappa 1 receptor antagonist norbinaltorphimine. In conclusion, the excitability of presynaptic nerve terminals in the chick ciliary ganglion can be modulated by the inhibition of at least three separate ion conductances following activation of kappa 1 opioid receptor sites in the nerve terminal region.

Spontaneous miniature hyperpolarizations of presynaptic nerve terminals in the chick ciliary ganglion.

Intracellular recordings from presynaptic nerve terminals in the chick ciliary ganglion revealed the presence of spontaneous miniature hyperpolarizations in virtually all (approximately 86%) nerve terminals examined. These spontaneous events appeared as small, brief hyperpolarizations at resting potential and were observed to increase or decrease as the membrane potential was depolarized or hyperpolarized from rest, respectively. The hyperpolarizing potentials were sensitive to blockade by tetraethylammonium and Ba2+, while caffeine increased then abolished these events. The voltage fluctuations were unaffected by tetrodotoxin, low Ca2+ external solution or the synaptic blockers, picrotoxin and strychnine. These spontaneous, transient, miniature hyperpolarizations may be due to the brief and co-ordinated activation of between 15-60 Ca(2+)-dependent K+ channels following the release of Ca2+ from internal stores.

An inward rectifier is present in presynaptic nerve terminals in the chick ciliary ganglion.

Inwardly rectifying voltage-sensitive channels have been detected in the cell bodies and axons of a number of excitable cells. The question of whether similar channels exist at axon terminals has been a matter of speculation for some time. We now report the first direct evidence for the existence of inward rectifiers in vertebrate presynaptic nerve terminals. Following impalement with intracellular electrodes, the large calyciform nerve terminals innervating chick ciliary ganglion neurons exhibit pronounced inward rectification upon hyperpolarization that increases with increasing current strength. The response is blocked by 2 mM Cs+, but is insensitive to Ba2+, tetraethylammonium and tetrodotoxin. The inward rectifier exhibits dependence on both Na+ and K+, but is unaffected by altering extracellular Ca2+. Ciliary neurons innervated by these nerve terminals display inward rectification with similar properties. We conclude that the inward rectifier present in these presynaptic nerve terminals resembles the H-current previously described in sensory ganglion neurons and the Q-current found in hippocampal pyramidal neurons. The presence of channels that are activated by hyperpolarization may serve to enhance the excitability of the calyciform nerve terminals, which are capable of relatively high frequencies (greater than 100 Hz) of discharge.

Results of mastectomy and postoperative irradiation in the management of locoregionally advanced carcinoma of the breast.

Between 1955 and 1984, 376 patients with locoregionally advanced breast carcinoma were treated at The University of Texas M. D. Anderson Cancer Center with mastectomy and irradiation and without adjuvant chemotherapy. Patients with inflammatory carcinoma or synchronous bilateral primary tumors were excluded. There were 202 patients with Stage IIIA disease and 174 patients with Stage IIIB disease (AJC Staging--1983). In 124 patients the surgical management was confined to the breast only--total mastectomy (BR) and in 252 dissection of the axilla was performed--extended total, modified radical, or classic radical mastectomy (BR + AX). All patients had postoperative irradiation. The follow-up period ranged between 8 and 34 years. At 10 years, the actuarial disease-specific, relapse-free survival (DSRFS) rate for the entire group was 40%, and the actuarial locoregional control rate was 82%. For patients with Stage IIIA disease the DSRFS was 48% and locoregional control rate was 88%. For those with Stage IIIB disease, the figures were 30% and 74%, respectively. Most of the failures occurred within 5 years of the mastectomy and essentially all occurred within 10 years. When analyzed by type of surgery, both the locoregional control and DSRFS rates were improved by the axillary dissection, the difference being largely caused by fewer axillary node recurrences after dissection of both the breast and axilla than after removal of the breast alone. In the 252 patients in whom the axilla was assessed, the number of positive nodes was a powerful predictor of both locoregional control and survival. The DSRFS rates at 10 years for patients with 0, 1-3, and greater than or equal to 4 positive nodes were 63%, 48%, and 30%, respectively. The actuarial locoregional control rates at 10 years exceeded 95% for patients with 0-3 positive nodes and 75% for those with greater than or equal to 4 nodes. These results show that locoregionally advanced breast cancer is not a uniformly fatal disease when treated without chemotherapy and provide a baseline upon which to assess the value of adjuvant systemic therapy for this stage of disease.

Hypofractionation: lessons from complications.

Hypofractionation refers to irradiation schemes with less than 5 fractions per week and larger doses per fraction than 2 Gy. It was initiated around 1960 to ease the burden for patients who had to come to the hospital to be treated every day and to save machine time. In the treatment of breast cancer at the University of Texas M.D. Anderson Cancer Center (UTMDACC) in 1962, the acute reactions of the skin were matched in 3- and 5-day treatment. More severe late sequelae were observed in the 3-day-a-week treatment than in the 5-day treatment. In 1966 the NSD formula was published and from thereon was used to adjust the total dose based on the number of fractions. Later, the TDF and CRE formulae were used. In almost all publications, a high percentage of severe complications after hypofractionation have been reported. There is overwhelming evidence that fraction size of more than 2 Gy produces late unfavorable sequelae, and therefore, despite the inconvenience for patients and the taxing of machine time, hypofractionation should not be used, unless there is a specific rationale concerning the tumor characteristics of doing so. In that case the total dose must be corrected in order not to increase the late morbidity.

Multimodal treatment for inflammatory breast cancer.

This is a retrospective study of 61 patients with clinically diagnosed breast cancer (IBC) treated with multimodality therapy between September 1977 and September 1985. All patients were scheduled to receive three courses of doxorubicin-based chemotherapy followed by mastectomy, further chemotherapy, and postoperative irradiation. Ten patients (16%) obtained a complete response, defined as either resolution of the clinical signs of inflammatory breast cancer (IBC) (4 patients) or no evidence of tumor in the mastectomy specimen (6 patients). Twenty-seven patients (45%) obtained a partial response, defined as a greater than 50% reduction in the clinical signs of inflammatory breast cancer. No response occurred in 24 patients (39%). Immediate mastectomy was done in 56 patients. Five patients whose disease was not resectable received preoperative irradiation. Nine patients at high risk for locoregional failure received postoperative irradiation immediately after mastectomy and before additional chemotherapy. Postoperative irradiation was given to the chest wall and peripheral lymphatics using standard or accelerated fractionation to a maximum dose of 60 Gy. Forty-six patients completed planned treatment including chemotherapy, surgery, and radiotherapy without failure. The minimum follow-up was 36 months. The 5-year actuarial disease-free survival was 70% for the complete response group, and 35% for the partial response group. All patients with no response failed by 34 months. The actuarial 5-year disease-free survival rate for the entire group was 27%. The 5-year actuarial locoregional control was 89% in the complete response group, 68% in the partial response group, 33% in the no response group, and 58% for all patients. Most failures were on the chest wall within the irradiated volume. Chest wall failures were more frequent in those who did not achieve brisk erythema or moist desquamation after postoperative irradiation. We conclude that multimodal treatment of patients with inflammatory breast cancer results in a low incidence of failure if complete response is obtained following initial chemotherapy. The locoregional control rate and actuarial 5-year disease-free survival for the entire group were not improved when mastectomy was done. Surgery should be done in those patients who respond adequately to chemotherapy, so that late sequelae of high-dose breast irradiation can be eliminated. Higher doses of postoperative irradiation may be required to improve local control in those patients with the poorest response to initial chemotherapy.

Long-range results for breast cancer patients treated by radical mastectomy and postoperative radiation without adjuvant chemotherapy: an update.

Between 1963 and 1977, 941 patients with carcinoma of the breast received, at the University of Texas M.D. Anderson Cancer Center, peripheral lymphatic irradiation alone or with chest wall irradiation after a radical or modified radical mastectomy. None of the patients received adjuvant chemotherapy. The incidence of patients with histologically involved axillary nodes was 70%. The lymphatics of the apex of the axilla, of the supraclavicular area, and of the internal mammary chain were irradiated in patients with histologically positive axillary nodes and/or in patients with central or inner quadrant primaries regardless of the axillary status. When in 1963 an electron beam became available, chest wall irradiation has been added to the peripheral lymphatics irradiation, primarily when there was a heavy infestation of the axillary nodes. The disease-free survival curves tend to flatten out at 10 years. At 10 and 20 years, the disease-free survival rates are respectively 55% and 50% for all patients, 44% and 40% for all patients with positive nodes, 56% and 48% for the patients with one to three positive nodes, and 33% and 30% for the patients with four or more positive nodes. The comparison of the mortality curves between the general population and the breast cancer patients seems to indicate a cured fraction, since the curves become parallel at 17 years. The highest incidence of failures is between 0 and 5 years, still a significant incidence between 5 and 10 years, but after 10 years the incidence of failures is relatively small.

Definitive radiotherapy for squamous cell carcinoma of the tonsillar fossa.

Between July 1968 and December 1983, 150 patients with previously untreated squamous cell carcinomas of the tonsillar fossa received megavoltage external beam irradiation with curative intent at U.T.M.D. Anderson Cancer Center. These patients were treated following a series of patients who had received radiotherapy between 1954 and May 1968. One hundred and thirty-seven patients were treated with conventional fractionation, the mean doses to the primary being 64.3 Gy, 67.8 Gy, 70.2 Gy, and 72.6 Gy for T1, T2, T3, and T4 lesions respectively. Thirteen patients were treated by altered fractionation schedules, 7 by hyperfractionation, and 6 by a concomitant boost to the primary. Elective bilateral neck irradiation was routine in all patients. A planned neck dissection was performed in 26 patients. The 5-year actuarial overall and disease-specific survival rates were 47% and 70%, respectively. Absolute local control rates with a minimum of 2 years follow-up after irradiation were 94%, 79%, 58%, and 50% for patients with T1, T2, T3, and T4 disease respectively. A total of 37 patients had local treatment failure; in 5 of 18 surgical salvage was successful. Only 4 patients with primary disease control developed failure in the neck and none of those with N0 or N1 disease did so when the primary was controlled. Twelve patients developed transient self-limited bone exposure, 7 developed osteoradionecrosis of the mandible, all requiring surgical resection. Most severe late complications occurred in patients with T3 and T4 lesions whose dose to the primary exceeded 67.5 Gy.

Differential effects of pallidal lesions on the behavioural responses to SKF 38393, LY 171555 and apomorphine in the rat.

The purpose of this study was to determine the role of the globus pallidus in the expression of dopamine D1- and D2-receptor mediated motor events. Rats were first injected stereotaxically with 6-hydroxydopamine in one medial forebrain bundle to denervate the ascending dopamine pathways in that hemisphere. Apomorphine and selective D1 and D2 agonists were then administered, at two dose levels, to establish characteristic response patterns. Subsequently the animals were given a secondary lesion by injecting kainic acid (0.2-1 microgram) into the ipsilateral globus pallidus and retested with the dopamine agonists over a period of two months. The kainate treatment itself caused spontaneous motor asymmetries, followed by aphagia, adipsia and hypersensitivity to touch. Contraversive circling, contralateral posture and grooming induced by systemic apomorphine were all abolished by the kainate treatment, whilst sniffing and head movements were facilitated. All activities induced by D1 stimulation were abolished or severely reduced under these conditions. By contrast, the contralateral posture and grooming elicited by D2 stimulation were spared, and only D2-dependent contraversive rotation, sniffing and head movements were reduced. All behavioural deficits were temporary and recovered partially or completely during the course of the experiment, but could not be overcome by increasing the dose of dopamine agonist. Post mortem histology revealed a consistent loss of pallidal neurons, together with more variable damage to extrapyramidal structures and the thalamus. The results show that all the D1-mediated, and certain of the D2-mediated motor responses depend on the integrity of the pallidum for their expression in the unilaterally 6-hydroxydopamine-treated rat.

T1-T2 squamous cell carcinoma of the glottic larynx treated with radiation therapy: relationship of dose-fractionation factors to local control and complications.

This is an analysis of 304 patients with invasive, previously untreated T1-T2 squamous cell carcinoma of the glottic larynx treated with radiation therapy at the University of Florida between October 1964 and December 1984. All patients had a minimum 2-year follow-up and 82% had at least 5 years of follow-up. Patients were excluded from the analysis of local control if they died within 2 years of treatment with the primary site continuously disease-free; all patients were included in the analysis of treatment complications. Patients were staged according to the 1983 AJCC system. Stage T2 was subdivided into 2 groups as follows: T2a (normal mobility) and T2b (decreased mobility). The rates of local control with radiation therapy were as follows: T1, 159/171 (93%); T2a, 50/65 (77%); and T2b, 31/43 (72%). Patients were further divided into subsets based on T stage and the surgical procedure that would have been required to resect the lesion. Local control was noted to improve with higher doses and dose per fraction in 4 of 7 subsets. In 2 of 3 subsets where a dose-fractionation relationship was not observed, there were no local recurrences in 1 subset and only 2 local recurrences in the other. The overall incidence of serious complications was 5/304 (1.6%) and was associated with T stage and with increasing total dose and dose per fraction.

Intracerebral SCH 23390 and catalepsy in the rat.

Stereotaxic injection of SCH 23390 (D-1 antagonist), but not water or ritanserin (5-HT antagonist), into all parts of the caudate-putamen, elicited an immediate and often long-lasting catalepsy in 79% of rats. Similar results were obtained with SCH 23390 delivered into the nucleus accumbens and globus pallidus, but not into a variety of other brain sites. The results support the idea that SCH 23390-induced catalepsy is related to the occlusion of dopamine D-1 receptors in the forebrain, particularly the striatum, although the topography of the catalepsy evoked by intrastriatal SCH 23390 did not match the distribution of D-1 sites labelled in autoradiographic studies.