Nicotinic receptor subtypes mediating relaxation of the normal human clasp and sling fibers of the upper gastric sphincter.

BACKGROUND: Proper function of the gastro-esophageal high pressure zone is essential for the integrity of the antireflux barrier. Mechanisms include tonic contractions and the decreased tone during transient lower esophageal sphincter relaxations. METHODS: We characterized the pharmacology of nicotinic receptors mediating relaxations of the human upper gastric sphincter (clasp and sling fibers) using currently available subtype selective nicotinic antagonists in tissue from organ transplant donors. Donors with either a history of gastro-esophageal reflux disease or histologic evidence of Barrett's esophagus were excluded. Clasp and sling muscle fiber strips were used for one of three paradigms. For paradigm 1, each strip was exposed to carbachol, washed, exposed to nicotinic antagonists then re-exposed to carbachol. In paradigm 2, strips were exposed to a near maximally effective bethanechol concentration then nicotine was added. Strips then were washed, exposed to nicotinic antagonists then re-exposed to bethanechol followed by nicotine. In paradigm 3, strips were exposed to bethanechol then choline or cytisine. KEY RESULTS: 100 µM methyllycaconitine has no inhibitory effects on relaxations, eliminating homomeric α7 subtypes. Subtypes composed of α4ß2 subunits are also eliminated because choline acts as an agonist and dihydro-beta-erythroidine is ineffective. CONCLUSIONS & INFERENCES: Because mecamylamine blocks the relaxations and both choline and cytisine act as agonists in both clasp and sling fibers, the nicotinic receptor subtypes responsible for these relaxations could be composed of α3ß4ß2, α2ß4, or α4ß4 subunits.

Enhanced nicotinic receptor mediated relaxations in gastroesophageal muscle fibers from Barrett's esophagus patients.

BACKGROUND: Increased nicotinic receptor mediated relaxation in the gastroesophageal antireflux barrier may be involved in the pathophysiology of reflux. This study is designed to determine whether the defects we previously identified in gastroesophageal reflux disease patients in- vivo are due to abnormalities of the gastric sling, gastric clasp, or lower esophageal circular (LEC) muscle fibers. METHODS: Muscle strips from whole stomachs and esophagi were obtained from 16 normal donors and 15 donors with histologically proven Barrett's esophagus. Contractile and relaxant responses of gastric sling, gastric clasp, or LEC fibers were determined to increasing concentrations of carbachol and to nicotine after inducing maximal contraction to bethanechol. Muscarinic receptor density was measured using subtype selective immunoprecipitation. KEY RESULTS: Barrett's esophagus gastric sling and LEC fibers have decreased carbachol-induced contractions. Barrett's esophagus sling fibers have decreased M2 -muscarinic receptors and LEC fibers have decreased M3 receptors. Relaxations of all three fiber types are greater in Barrett's esophagus specimens to both high carbachol concentrations and to nicotine following bethanechol precontraction. The maximal response to bethanechol is greater in Barrett esophagus sling and LEC fibers. CONCLUSIONS & INFERENCES: The increased contractile response to bethanechol in Barrett's specimens indicates that the defect is likely not due to the smooth muscle itself. The enhanced nicotinic receptor mediated response may be involved in greater relaxation of the muscles within the high-pressure zone of the gastroesophageal junction during transient lower esophageal sphincter relaxations and during deglutitive inhibition and may be involved in the pathophysiology of gastroesophageal reflux disease.

Characterization of the distal esophagus high-pressure zone with manometry, ultrasound and micro-computed tomography.

BACKGROUND: We sought to determine how the individual components of the distal esophagus and proximal stomach form the gastroesophageal junction high-pressure zone (GEJHPZ) antireflux barrier. METHODS: An endoscopic ultrasound/manometry catheter was pulled through the proximal stomach and distal esophagus in 20 normal subjects. The axial length and width of individual structures on endoscopic ultrasound were measured. The anatomic orientation of gastroesophageal junction (GEJ) components was examined in two organ donor specimens using micro-computed tomography (micro-CT). KEY RESULTS: The three distinct structures identified within the GEJHPZ, from distal to proximal, were as follows: the gastric clasp and sling muscle fiber complex, crural diaphragm, and lower esophageal circular smooth muscle fibers (LEC). The LEC was statistically significantly thicker than adjacent esophageal muscles. These structures were associated with three pressure peaks. The pressure peak produced by the clasp/sling fiber complex often overlapped with the pressure peak from the crural diaphragm. The most proximal peak, associated with the LEC, was significantly greater and bimodal in nine of 20 subjects. This bimodal LEC pressure peak correlated with two areas of thickened muscle observed with ultrasound. Micro-CT of GEJ from organ donors confirmed the two areas of thickened muscle. CONCLUSIONS & INFERENCES: Three distinct anatomic structures, the clasp and sling muscle fibers, crural diaphragm, and LEC combine to form the antireflux barrier of the proximal stomach and distal esophagus. The clasp and sling muscle fibers combine with the crural diaphragm to form a distal pressure profile. The more proximal LEC has a bimodal pressure profile in some patients.

Regulation of bladder muscarinic receptor subtypes by experimental pathologies.

1 The M3 muscarinic receptor subtype is widely accepted as the receptor on smooth muscle cells that mediates cholinergic contraction of the normal urinary bladder and other smooth muscle tissues, however, we have found that the M2 receptor participates in contraction under certain abnormal conditions. The aim of this study was to determine the effects of various experimental pathologies on the muscarinic receptor subtype mediating urinary bladder contraction. 2 Experimental pathologies resulting in bladder hypertrophy (denervation and outlet obstruction) result in an up-regulation of bladder M2 receptors and a change in the receptor subtype mediating contraction from M3 towards M2. Preventing the denervation-induced bladder hypertrophy by urinary diversion prevents this shift in contractile phenotype indicating that hypertrophy is responsible as opposed to denervation per se. 3 The hypertrophy-induced increase in M2 receptor density and contractile response is accompanied by an increase in the tissue concentrations of mRNA coding for the M2 receptor subtype, however, M3 receptor protein density does not correlate with changes in M3 receptor tissue mRNA concentrations across different experimental pathologies. 4 This shift in contractile phenotype from M3 towards M2 subtype is also observed in aged male Sprague-Dawley rats but not females or either sex of the Fisher344 strain of rats. 5 Four repeated, sequential agonist concentration response curves also cause this shift in contractile phenotype in normal rat bladder strips in vitro, as evidenced by a decrease in the affinity of the M3 selective antagonist p-fluoro-hexahydro-sila-diphenidol (p-F-HHSiD). 6 A similar decrease in the contractile affinity of M3 selective antagonists (darifenacin and p-F-HHSiD) is also observed in bladder specimens from patients with neurogenic bladder as well as certain organ transplant donors. 7 It is concluded that although the M3 receptor subtype predominantly mediates contraction under normal circumstances, the M2 receptor subtype can take over a contractile role when the M3 subtype becomes inactivated by, for example, repeated agonist exposures or bladder hypertrophy. This finding has substantial implications for the clinical treatment of abnormal bladder contractions.

Immune function, mitogenicity, and angiogenic growth factor concentrations in lean and obese rodent sera: implications in obesity-related prostate tumor biology.

Some studies suggest that several tumors have a greater incidence in those patients with a high fat diet, such as colon, breast, and prostate. However, we wanted to determine the effects of obesity alone, independent of diet, on the progression of prostate tumor growth. Using a genetic model of obese and lean Zucker rats, we wanted to demonstrate any sera differences in the concentration of basic fibroblast growth factor (FGF-2) and vascular endothelial cell growth factor (VEGF), two important factors involved in the growth and progression of prostate cancer. We also wanted to investigate if there were any differences in immune function between the two sera, which could also account for uninhibited tumor growth, as well as differences in mitogenic stimulation. Female Zucker rat obese and lean sera were analyzed using ELISA assays for FGF-2, VEGF, and macrophage inflammatory protein-1 alpha (MIP-1a), as a measure of macrophage function. In addition, the sera of lean and obese sera were plated on wells growing LNCaP prostate cancer cells to determine differences in mitogenicity. We found a greater concentration of FGF-2 in the sera from obese Zucker rats compared to lean Zucker rats: 6.32+/-0.56 vs 3.48+/-0.34 pg/ml, respectively, P<0.05). We also demonstrated a greater concentration of VEGF in obese rat sera compared to lean sera: 54.4+/-4.1 vs 38.0+/-2.9 pg/mL, respectively, P<0.05). We detected a trend in mitogenic stimulation among LNCaP cells along the higher concentrations of the dose-response curve (0.72+/-0.06 vs 0.51+/-0.5). However, this was not statistically significant. In addition, we did not find a significant difference in MIP-1a macrophage activity levels between sera. To conclude, we speculate that the greater concentrations of VEGF and FGF-2 in the sera of obese rodents vs lean rodents may account for some of the differences seen in obesity-related tumor growth seen in the human condition. However, the lack of any sera differences of immune function, as measured by macrophage activity, as well as no significant differences on mitogenic proliferation on LNCaP prostate cancer cells, suggests that other mechanisms may exist to explain differences seen in obesity-related prostate tumor biology.

Impaired venous hemodynamics in a minority of patients with chronic leg ulcers due to sickle cell anemia.

BACKGROUND: Chronic or recurrent leg ulceration occurs in 25% of sickle cell anemia patients, but not in the remaining 75%. Doppler studies of venous function were normal in 16 sickle cell anemia patients with leg ulcers. PATIENTS AND METHODS: Venous Duplex Ultrasound was used to study 33 sickle cell anemia patients with chronic leg ulcers. RESULTS: Six of the 33 patients had venous reflux in at least one leg. CONCLUSIONS: Venous insufficiency may contribute to the development of leg ulcers in a minority of sickle cell anemia patients. A minority of sickle cell anemia patients with chronic leg ulcers can be shown to have leg venous reflux by duplex ultrasound imaging.

Onycholysis as a complication of systemic chemotherapy: report of five cases associated with prolonged weekly paclitaxel therapy and review of the literature.

BACKGROUND: Onycholysis has been reported in association with the use of several noncytotoxic drugs and with chemotherapy in 135 patients. Onycholysis may be precipitated by exposure to ultraviolet radiation. METHODS: The authors studied 91 patients who received paclitaxel and 187 patients who received doxorubicin. RESULTS: Onycholysis occurred in 5 of 21 patients who received > 6 courses of weekly paclitaxel, developing in the summer months in all 5 patients. It did not occur in patients who received fewer weekly paclitaxel courses or those who were treated every 3 weeks. Onycholysis did not occur in 187 patients who received doxorubicin. Review of the literature revealed that onycholysis is nearly exclusively associated with anthracycline and taxane therapy. CONCLUSIONS: Prolonged weekly paclitaxel, other taxanes, and anthracyclines cause onycholysis in some patients, which may be precipitated by exposure to sunlight. Patients receiving these drugs should protect their nails from sunlight.

Bilateral pleural effusions in a beta-thalassemia intermedia patient with posterior mediastinal extramedullary hematopoietic masses.

Intractable bilateral exudative pleural effusions developed, following systemic sepsis without pulmonary infection, in a beta-thalassemia intermedia patient with longstanding mediastinal hematopoietic masses. The pleura were not infiltrated by hematopoietic cells. Bilateral talc pleurodesis successfully controlled the effusions.

Selective alkylation of rat urinary bladder muscarinic receptors with 4-DAMP mustard reveals a contractile function for the M2 muscarinic receptor.

Our previous data indicate that M3 muscarinic receptors mediate carbachol induced bladder contractions. The data presented here were obtained by selective alkylation of M3 receptors with 4-DAMP mustard and suggest that the M2 receptor subtype may be involved in inhibition of beta-adrenergic receptor induced relaxation, therefore, allowing recontraction. Alkylation resulted in 85% of M3 receptors and 65% of M2 receptors unable to bind radioligand as demonstrated by subtype selective immunoprecipitation. Rat bladder strips subjected to our alkylation procedure contracted submaximally, and direct carbachol contractions were inhibited by antagonists with affinities consistent with M3 receptor mediated contraction. In contrast, the affinities of antagonists for inhibition of carbachol induced recontractions following isoproterenol stimulated relaxation in the presence of 90 mM KCl, indicated a contractile function for the M2 receptor that was not observed in control strips. In conclusion, these studies demonstrate a possible role for the M2 subtype in bladder smooth muscle contraction.

M2 muscarinic receptor contributes to contraction of the denervated rat urinary bladder.

In vitro bladder contractions in response to cumulative carbachol doses were measured in the presence of selective muscarinic antagonists from rats that had their major pelvic ganglion bilaterally removed. Denervation induced both hypertrophy and a supersensitivity of the bladders to agonist. The affinities in control bladders for antagonism of carbachol-induced contractions were consistent with M3-mediated contractions. Affinities in denervated bladders for 4-diphenlacetoxy-N-methylpiperidine methiodide (8.5) and p-fluoro hexahydrosilodifenidol (6.6) were consistent with M2-mediated contractions, although the methoctramine affinity (6.5) was consistent with M3-mediated contractions. Subtype-selective immunoprecipitation of muscarinic receptors revealed a 50% increase in total and a 60% increase in M2 receptor density with no change in M3 receptor density in denervated bladders compared with normal or sham-operated controls. This increase in M2 receptor density is consistent with the change in affinity of the antagonists for inhibition of carbachol-induced contractions and may indicate that M2 receptors or a combination of M2 and M3 receptors directly mediates smooth muscle contraction in the denervated bladder.

Characterization of muscarinic cholinergic receptor subtypes in rat prostate.

The purpose of this study was to characterize the muscarinic receptor subtypes in the individual lobes of the rat prostate. Immunoprecipitation was performed on homogenates of these 3 lobes using antibodies to the m1-m4 muscarinic receptor subtypes. Reverse transcriptase polymerase chain reaction assays (RT-PCR) were also performed using primers specific for each of the five muscarinic receptor subtypes (m1-m5). The susceptibility of the receptors to degradation by endogenous prostate proteases was assessed by mixing rat ventral prostate with rat heart (m2) and rat parotid (m3) prior to immunoprecipitation. In the ventral lobe, transcripts for the m1-m4 subtypes were amplified whereas in the dorsal and lateral lobes only the m2 and m3 sets of primers amplified PCR products of the predicted size. Immunoprecipitation of the ventral lobe resulted in predominantly m3 receptors, while the majority of receptors immunoprecipitated from lateral and dorsal lobes were the m2 subtype. The m3 muscarinic subtype was apparently susceptible to degradation by prostate proteases whereas the m2 subtype was not. These results demonstrate a regional distribution in the subtypes of muscarinic receptors in the rat prostate, and a greater susceptibility of the m3 receptor to degradation during immunoprecipitation than the m2 subtype.

Prejunctional M1 facilitory and M2 inhibitory muscarinic receptors mediate rat bladder contractility.

Subtype-selective muscarinic antagonists effects on carbachol-induced and electric field-stimulated contractility of rat bladder were compared in vitro. Schild plot analysis of cumulative carbachol dose-response curves in the presence of antagonists was consistent with M3-mediated bladder contractions. However, nerve-evoked contractions were inhibited 15% at 30 Hz (P < 0.01) by 10 nM pirenzepine (M1-selective antagonist), whereas 10 nM methoctramine (M2-selective antagonist) increased these contractions by 17% at 30 Hz (P < 0.01). Identical doses had no effect on carbachol-induced contractions, indicating prejunctional M1 facilitory and M2 inhibitory receptors. m1 Receptors could not be identified by subtype-selective antibodies, nor could the m1 transcript be identified by Northern hybridization. However, m1, m2, m3, and m4 transcripts were identified in rat bladder using the reverse transcriptase-polymerase chain reaction, providing support for the existence of the m1 subtype. In conclusion, strong evidence is provided for the existence of prejunctional M1 facilitory and M2 inhibitory and postjunctional M3 receptors modulating contractility in the rat urinary bladder.

Chemotherapy is a safe and effective initial therapy for infected malignant breast and chest wall ulcers.

BACKGROUND AND OBJECTIVES: Locally advanced breast cancers may form large, infected skin ulcers, which were traditionally treated with radiation therapy. Neoadjuvant chemotherapy is now standard treatment for locally advanced breast cancer. METHODS: The response of 33 patients with ulcerated breast cancer to primary chemotherapy was retrospectively analyzed. Antibiotics were not used in primary treatment. Tumor and ulcer responses were evaluated independently. RESULTS: Chemotherapy alone healed 18 of these ulcers. Neither responding nor refractory patients developed sepsis during this treatment. CONCLUSIONS: Chemotherapy is safe and effective treatment for patients with infected malignant breast ulcers and does not cause systemic sepsis.

Estrogen receptor immunocytochemistry in paraffin embedded tissues with ER1D5 predicts breast cancer endocrine response more accurately than H222Sp gamma in frozen sections or cytosol-based ligand-binding assays.

BACKGROUND: Historically, estrogen receptor (ER) determinations have been made by the ligand-binding assay of tumor homogenates, primarily by the dextran-coated charcoal method (DCC). Immunocytochemical assays (ICA) for ER are more recent and have been executed mostly on frozen sections with the monoclonal antibody H222Sp gamma (H222). Lately, new monoclonal antibodies derived by recombinant ER technology have been developed that work well on paraffin embedded, formalin fixed tissue sections. However, there is little information as to whether such assays prognosticate endocrine response. METHODS: Using antigen retrieval, the immunoglobulin G1 monoclonal antibody ER1D5, and the streptavidin-biotin detection system, 74 patients with breast cancer in whom endocrine response was known were assayed and the results compared with ER by DCC and ER by ICA in frozen section with H222. RESULTS: ER1D5 in paraffin provided the highest correlation with endocrine response (Kendall's tau [r] = 0.57; P<0.001) whereas ER by DCC failed to correlate (r= -0.002; P<0.99). ER1D5 in paraffin correlated weakly though significantly with DCC (Kappa Statistic [K] = 0.204; P<0.02). H222 in frozen sections also correlated moderately with endocrine response (r = 0.34; P<0.001). CONCLUSIONS: ER can be detected in routine tissue sections processed with antigen retrieval and ER1D5, and can be relied upon to provide accurate prognostic information regarding response to endocrine therapies in breast cancer patients.

Estrogen receptor immunocytochemistry: the promise and the perils.

Estrogen receptor immunocytochemistry (ERICA) is favored over dextran-coated charcoal (DCC) or sucrose gradient assay (SGA) by many pathologists and oncologists since it allows an estimation of tumor cell and tissue heterogeneity and permits assays to be performed on specimens not suitable for DCC/SGA. Additionally, ERICA can be performed with greater ease and with less expense at the level of the community hospital pathology laboratory. Initially, like DCC/SGA, ERICA had to be done on fresh or frozen tumor samples or face a significant loss in sensitivity when applied to formalin-fixed, paraffin-embedded sections. Recently, several anti-estrogen receptor (ER) antibodies have appeared which can be successfully employed to assay routinely prepared tissue sections if used in conjunction with new antigen-retrieval techniques such as the microwave oven and citrate buffers. However, more work is needed to correlate results of these new procedures with biochemical ER assays, endocrine response, and survival before they can be reliably employed as prognostic parameters. Furthermore, if any ER assay is to be useful and valid, strict attention must be paid to details of specimen collection, freezing, and fixation in order to inhibit receptor degradation and false negative results.