Recent Submissions

  • The Effect of Buccal versus Vaginal Misoprostol on Time to Favorable Simplified Bishop Score

    Rice Thompson, Sonya; Allen, Jennifer (2020-06)
    OBJECTIVE: To evaluate whether buccal or vaginal Misoprostol provides a statistically significant shorter time interval to a favorable simplified Bishop score, and to determine if there is a significant difference in time to delivery between these two modes of administration. DESIGN: Retrospective cohort study via chart review SETTING: Augusta University, Augusta, Georgia PATIENTS: Mothers who delivered full term singleton gestations after being admitted for induction of labor at Augusta University from January 1, 2015 to July 31, 2018 with an initially unfavorable simplified Bishop score on cervical exam. Exclusion criteria: (1) Favorable initial simplified Bishop score (>5) at the time of onset of induction of labor, (2) multiple gestation, (3), pre-term gestation and (4) use of multiple different modalities for cervical ripening. INTERVENTION: Medical records were reviewed to determine if mothers meeting the criteria above had a statistically significant difference in time to favorable simplified Bishop score using vaginal versus buccal Misoprostol and/or a statistically significant difference in time to delivery. MAIN OUTCOME MEASURE: The primary outcome was time to favorable simplified Bishop score (>5). The secondary outcome was time to delivery. RESULTS: • Primary outcome- no significant difference between the buccal and vaginal groups in median time from initial Misoprostol dose to time of favorable simplified Bishop score (p=0.371). • Secondary outcome- no significant difference between the buccal and vaginal groups in median time from initial Misoprostol dose to time of delivery (p=0.371). • No significant difference in complication rate between the buccal and vaginal groups (p=0.199).
  • Ectopic Pregnancy Interactive Model

    Fambrough, J; Allen, J; Latif, E; Edmondson, A; Department of Obstetrics and Gynecology (2017-03)
    Ectopic pregnancy accounts for 2% of all first-trimester pregnancies, and it is the number one cause of maternal death in the first trimester. Women present with ectopic pregnancies across the nation, and it is imperative for future healthcare providers to identify these patients, rule out other diagnoses, and treat them in a time sensitive manner. Most educational materials regarding ectopic pregnancy are limited to textbooks and printed literature, yet the current generation of healthcare students is more responsive to interactive, feedback-driven, learning options. Studies regarding medical student education show that motivational processes are undervalued in curriculum development and stimulating motivation can influence outcomes of curriculum performance. It has been shown that intrinsic motivation (i.e., learning for the sake of learning and patients) leads to better performance and well being. Interactive modules enable students to make decisions without the repercussions of an incorrect answer, and they are encouraged to actively learn from mistakes and successes.
  • The Effects of Exclusively Rooming-In on Breastfeeding Rates

    Titus, Hamer M.; Raines, Anna; Looney, Stephen; Fambrough, Jada; Diamond, Michael P.; Allen, Jennifer T.; Department of Obstetrics and Gynecology (2019-09)
    OBJECTIVE: To evaluate whether the rooming-in model of postpartum care has improved breastfeeding rates at Augusta University Medical Center. METHODS: In this retrospective cohort study, we reviewed the medical records of 302 mother-baby pairs before exclusively rooming in (BERI) and 305 pairs after exclusively rooming in (AERI) to determine rooming-in’s effect on breastfeeding rates. The primary outcome was inpatient breastfeeding rates, while the secondary outcome was breastfeeding rates at six weeks postpartum. RESULTS: The data shows a significant decrease in inpatient breastfeeding rates from 59.6% BERI to 48.9% AERI (p=0.028), but a significant increase in six-week exclusive breastfeeding rates from 17.0% BERI to 32.4% AERI (p=0.017). Black mothers and single mothers show a decrease in inpatient breastfeeding rates (39.8% to 31.7% and 36.7% to 34.4%, respectively), but an increase in 6-week breastfeeding rates (8.2% to 13.6% and 5.4% to 16.6%, respectively) from BERI to AERI. White mothers and married mothers show little difference in inpatient breastfeeding rates (75.6% to 73.3% and 73.5% to 72.3%, respectively), but a significant increase in 6-week breastfeeding rates (37.0% to 51.7% and 38.2% to 53.9%, respectively) from BERI to AERI. CONCLUSION: This study shows that overall, exclusively rooming-in did not improve immediate breastfeeding rates postpartum, but did improve six-week postpartum breastfeeding rates. Stratification by race and marital status shows that these are two variable that have an effect modification on breastfeeding rates with regards to rooming-in.
  • Predicting Violence Risk and Recidivism in Female Parolees: A State-Wide Sample

    Britt, Jessica Y.; Patton, Christina L.; Remaker, Dominique N.; Vitacco, Michael J.; Prell, Lettie; Department of Psychiatry (2020-07-20)
  • Cultural humility in internship training: Beyond checking the box

    Britt-Thomas, Jessica Y.; Department of Psychiatry (2022-07-20)
    Preparing psychology interns for practice in forensic psychology requires deep consideration of cultural factors. This cannot be accomplished by embedding a "cultural discussion" into a didactic to check a box for required trainings; internships must cultivate an environment that encourages trainees and supervisors alike to examine and question how our own identities factor into our daily decisions and interactions. Cultural humility requires self-reflection of one’s cultural identities as it relates to others, including privileged and oppressed identities. Having such discussions early and often during the internship year can establish expectations and build a culture of reflection, openness, and ongoing growth.
  • Comorbid Substance Abuse and Mental Illness on Impulsivity

    Patel, Kajol K.; McEvoy, Joseph P.; Miller, Brian J.; Britt, Jessica Y.; Department of Psychiatry (2022-07-20)
    Introduction: Impulsivity, or a lack of self-control, has been identified as a significant risk factor in individuals with substance abuse. Several studies have shown that the impact of impulsivity affects the onset of substance abuse, relapsing substance abuse, and outcomes during substance abuse treatment. Impulsivity has also been defined as a trait characteristic in multiple psychiatric disorders (i.e. schizophrenia, depression). Furthermore, studies have identified impulsivity as a mediating factor between psychiatric disorders and mental illness. Methods: Eligible participants were identified by practitioners at the Augusta University Psychiatry and Health Behaviors outpatient clinic and Serenity Behavioral Health Systems (n=47). Participants were administered the UPPS-short and BIS-11 scales via phone interviews. Follow-up phone interviews were conducted 30 days after the initial interview to establish test-retest validity. Of those that completed the initial assessment, 31 participants completed the follow-up assessment. Results: When comparing the UPPS and BIS scores in substance abusers and non-substance abusers, scores were higher in the substance abuse group compared to the non-substance abuse group, although this difference did not achieve significance (p = 0.19 and p = 0.43, respectively). UPPS and BIS scores correlated significantly with each other at initial assessment (r=0.79, p<0.001) and follow-up (r=0.82, p<0.001). The initial assessments of the UPPS and BIS also correlated significant with the follow-up assessments (r=0.74, p<0.001 and r=0.83, p<0.001, respectively). Conclusion: Results of the study indicate that impulsivity was higher in the substance abusing sample compared to the non-substance abusing sample, although significance was not reached. A decreased p-value in the entire sample as compared to previous analyses performed on a partial sample suggests that the current sample lacks power. Increased sample size may allow for the analyses to reach significance.
  • The Mentoring Minute Compendium

    Stepleman, Lara; Liang, Yan; Coleman, Taylor; Nall, Heather; Williams, Matthew; Samuels, Stevauney; Zimmerman, Danielle; Medical College of Georgia (2022-07-20)
    A collection of brief overviews of evidence-based mentoring practices
  • Intro to Anatomic Imaging

    Tally, Toby C (2021-08-12)
  • EFFECTS OF SODIUM BICARBONATE ON GLUCOSE HOMEOSTASIS AND BLOOD PRESSURE IN CHRONIC KIDNEY DISEASE

    Mannon, Elinor; Department of Philosophy (Augusta University, 2021-10)
    Sodium bicarbonate (NaHCO3) is a therapeutic used in chronic kidney disease (CKD). NaHCO3 is typically used to treat metabolic acidosis, but clinical studies have indicated that NaHCO3 supplementation may slow CKD progression. As such, NaHCO3 is now given to patients with CKD to slow the decline of glomerular filtration rate. However, the consequences of chronic NaHCO3 supplementation in CKD remain unclear. Acidosis has been associated with insulin resistance, and correction of acidosis with NaHCO3 was reported to improve insulin sensitivity. Our goal in Aim 1 was to determine whether acid and alkali loading would promote loss of acid-base homeostasis and consequently decrease insulin sensitivity. We determined that the blood glucose response to insulin is enhanced following renal mass reduction, and that this response is not reversed by an acidosis. Additionally, the development of an alkalosis did not impair the blood glucose response to insulin. Alkali can promote potassium (K+) wasting, and an association between K+ wasting and insulin resistance has been identified in clinical and basic science research. Our goal in Aim 2 was to identify whether chronic NaHCO3 treatment may promote loss of insulin sensitivity through effects on K+ status. We determined that chronic NaHCO3 treatment impairs insulin sensitivity when combined with other K+ wasting stimuli. K+ deprivation alone also impaired the blood glucose response to insulin, however these impairments in insulin sensitivity were not directly related to decreases in intracellular [K+]. Salt-sensitivity increases as functional renal mass declines, and chronic sodium (Na+) loading with NaHCO3 may contribute to hypertension in patients with CKD. Our goal in Aim 3 was to investigate whether NaHCO3 loading promotes similar levels of Na+ and volume retention, and hypertension as sodium chloride (NaCl) loading does in a rat model of CKD. We found that NaHCO3 was pro-hypertensive, but to a lesser degree than NaCl, despite similar amounts of Na+ and volume retention. From these studies we concluded that NaHCO3 does not improve insulin sensitivity through its effects on acid-base status. Further, access to dietary K+ may improve insulin sensitivity with chronic NaHCO3 treatment. Finally, NaHCO3 can promote hypertension in CKD.
  • A NOVEL NETWORK BASED LINEAR MODEL FOR ENRICHMENT OF SYNERGISTIC DRUG COMBINATIONS

    Li, Jiaqi; Department of Physiology (Augusta University, 2021-07)
    Drug combination therapies can improve drug efficacy, reduce drug dosage, and overcome drug resistance with respect to cancer treatments. Current research strategies to determine which drug combinations have a synergistic effect rely mainly on clinical or empirical experience and screening predefined pools of drugs. Given the number of possible drug combinations, the speed and scope to find new drug combinations are very limited using these methods. Due to the exponential growth in these combinatorials, it is difficult to test all possible outcomes in the lab. Several large-scale public genomic and phenotypic resources that provide data from single drug-treated cells as well as data from small molecules deliver a wealth of cellular response information. This data gives opportunity to overcome limitations of the current methods. The development of a new strategy for advanced data processing and analysis that includes a computational prediction algorithm is highly desirable. Because of this, a program was written that predicts synergistic drug combinations using gene regulatory network knowledge and an operational module unit (OMU) system generated from single drug genomic and phenotypic data. As a proof of principle, we applied the pipeline to a group of anticancer drugs and demonstrated how the algorithm could help researchers efficiently find possible synergistic drug combinations using single drug data to evaluate all possible drug pairs.
  • MODELING THE SIMULTANEOUS EFFECTS OF COPY NUMBER VARIATION AND METHYLATION ON GENE EXPRESSION USING NEXT GENERATION SEQUENCING DATA

    Claussen, Henry; Department of Physiology (Augusta University, 2021-07)
    The collection and order of nucleobases in a strand of DNA, called the primary sequence, is one of the most important pieces of information in the study of the human body. The proteins which regulate all biological functions in the body are synthesized based on the structure of the DNA molecule. The next generation sequencing (NGS) process of sequencing RNA transcripts, known as RNA-seq, has become a powerful alternative to traditional microarray technology. NGS is used to measure the levels of gene expression, detect structural DNA variations from the human reference genome, and uncover the epigenetic modifications of methylation. Despite its prevalence in genetic research, RNA-seq data suffers from the statistical complication known as ”large p small n” where the predictor variables greatly outnumber the subjects in a study. In this research we propose combining all three types of data into a multivariate linear model. With the implementation of a variable selection process for preliminary dimension reduction and the application of a Group LASSOapproach, we hope to reduce the complexity and dimension of NGS data to a manageable and, most importantly, interpretable level. Changes in gene expression levels have been linked with the development of harmful diseases such as cancer. A successful model will provide insight on the simultaneous effects that methylation and structural variation have on gene expression in the body.
  • Predictive Inference for Linear and Circular Concomitants with Biomedical Applications

    Howie, Melissa; Department of Philosophy (Augusta University, 2021-07)
    Let (X_i, Y_i), for i=1,...,n, be a random sample from a bivariate distribution. If the sample is ordered with respect to one of the variables, say X, then the rth ordered X-value is called the rth order statistic and is denoted X_{r:n}. The Y-value corresponding to this value is called the concomitant of the rth order statistic and is denoted Y_{[r:n]}. In biomedical research, there is an interest in predicting the concomitant variable corresponding to the rth order statistic of the other variable. For example, one may be interested in predicting the time at which a patient has the peak blood pressure or the mercury level in fish where the water is most polluted. One such distribution of interest is the bivariate exponential conditionals distribution (BEC), whose conditional distributions are both exponential. The asymptotic predictive distribution of the concomitants of order statistics from the BEC is derived. The results are used in a prediction problem involving the mercury concentration in largemouth bass sampled from Florida lakes, as a function of surface water pollution level. Clinicians are often confronted with data such that one variable is linear and the other variable is circular, i.e., measured as an angle. A particular linear-circular distribution of interest is the exponential circular normal distribution. The predictive distribution of concomitants of order statistics from the exponential circular normal distribution is derived. The results are applied to predicting the future value of time at maximum heart rate in subjects from the Augusta Heart Study, a longitudinal study of normotensive children with verified family histories of cardiovascular diseases (e.g., hypertension and premature myocardial infarction).
  • DEFINING THE ROLE OF AQUEOUS HUMOR PROTEOME IN GLAUCOMATOUS OPTIC NEUROPATHY

    Kodeboyina, Sai Karthik; Department of Physiology (Augusta University, 2021-05)
    Aqueous humor (AH) is a fluid in the anterior and posterior chambers of the eye that contains proteins associated with vision disorders including glaucoma. We performed comprehensive characterization of AH proteins and evaluated their association with optic nerve and visual field changes in glaucoma patients. AH reference database was developed to include proteomic and clinical information from cataract and glaucoma patients. Aqueous humor samples from 251 cataract and glaucoma patients were analyzed using Liquid-Chromatography Mass spectrometry (LC-MS/MS). Retinal nerve fiber layer (RNFL) thickness was evaluated with the SPECTRALIS Tracking Laser Tomography. Optic nerve head imaging was performed using Heidelberg Retinal Tomograph (HRT). Visual fields were analyzed with the Humphrey Visual Field analyzer. Statistical analyses were performed to discover the relationship between AH proteins and demographic characteristics, RNFL, optic nerve, and visual field parameters. AH reference database and website was developed using standard software tools including Visual Studio, ASP.NET, SQL, C#, and HTML. A total of 1774 unique proteins were identified in 251 AH samples of which 233 proteins were expressed in at least half of samples. Five protein families were discovered including apolipoproteins, complements, immunoglobulins, serine protease inhibitors (SERPINS) and insulin like growth factors (IGF). A total of 38 proteins significantly correlated with at least one RNFL thickness measure including average, inferior and superior thicknesses. Similarly, 62 proteins significantly associated with at least one HRT parameter such as cup shape measure, cup-disc area ratio and rim area. A total of 11, 9, 7, and 6 proteins were significantly correlated with pattern standard deviation, visual field index, mean deviation, and glaucoma hemifield test respectively. Strongly associated proteins include APOD, APOH, C4A, C4B, C7, IKHV3-9, IGKV2-28, SERPINA1, SERPINF1, IGFBP6, and IGFBP7. They are involved in immune responses, signaling, binding, and metabolic functions. These findings provide targets for future studies investigating molecular mechanisms and new therapies for glaucoma. Moreover, the database would serve as a resource for researchers pursuing AH proteomic and glaucoma studies.
  • Clinical Skills 1 MCG Competency-Based Direct Observation Master Rubric

    Bond, M; Department of Internal Medicine (Augusta University, 2021-05-19)
    Year 1 Clinical Skills grading rubric for directly observed standardized patient encounters involving communication skills, history taking, and physical exam components
  • Clinical Skills 2 Performance Evaluation

    Bond, M; Department of Internal Medicine (Augusta University, 2021-08-19)
    Year 2 Clinical Skills MCG Competency-Based Direct Observation tool for use in hospital based encounters.
  • OPTIMIZED ISOLATION AND QUANTIFICATION OF IN VIVO DISTRIBUTION OF EXOSOMES FOR POTENTIAL TARGETED THERANOSTIC APPLICATION

    Rashid, Mohammad Harun; Department of Biochemistry and Molecular Biology (Augusta University, 2019-07)
    Exosomes are critical mediators of intercellular crosstalk and regulators of the cellular/tumor microenvironment. Exosomes have great prospects for clinical application as a theranostic and prognostic probe. Nevertheless, the advancement of exosome research has been thwarted by our limited knowledge of the most efficient isolation method and the in vivo trafficking. Here we have shown that a combination of two size-based methods using a 0.20 μm syringe filter and 100k centrifuge membrane filter followed by ultracentrifugation yields a greater number of uniform exosomes compared to other available methods. We demonstrated the visual representation and quantification of the differential in vivo distribution of radioisotope 131I-labeled exosomes from diverse cellular origins, e.g., tumor cells with or without treatments, myeloid-derived suppressor cells and endothelial progenitor cells. We also determined that the distribution was dependent on the exosomal protein/cytokine contents. Further, we also generated engineered exosomes expressing precision peptide for targeting CD206 positive M2-macrophages. M2-macrophages participate in immune suppression, epithelial to mesenchymal transition, invasion, angiogenesis, tumor progression and subsequent metastasis foci formation. Given their pro-tumorigenic function and prevalence in most malignant tumors with lower survival, early in vivo detection and intervention of M2-macrophages may boost the clinical outcome. To determine in vivo distribution of M2-macrophages, we adopted 111In-oxine based radiolabeling of the targeted exosomes and SPECT. When injected these radiolabeled targeted exosomes into 4T1 breast tumor-bearing mice, exosomes accumulated at the periphery of the primary tumor, metastatic foci in the lungs, in the spleen, and liver. Ex vivo quantification of radioactivity also showed similar distribution. Injected DiI dye-labeled exosomes into the same mice showed the adherence of exosomes to the CD206 positive macrophages on ex vivo fluorescent microscopy imaging, confirming the targeting efficacy of the exosomes. In addition, we utilized these engineered exosomes to carry the Fc portion of mouse IgG2b with the intention of augmenting antibody-dependent cell-mediated cytotoxicity. We have auspiciously demonstrated that M2-macrophage targeting therapeutic exosomes deplete M2-macrophages both in vitro and in vivo, and reduce tumor burden in a metastatic breast cancer model. The applied in vivo imaging modalities can be utilized to monitor disease progression, metastasis, and exosome-based targeted therapy.
  • THE ROLE OF GPR109A IN REGULATION OF RETINAL ANGIOGENESIS AND BLOOD-RETINAL BARRIER AS A POTENTIAL THERAPEUTIC TARGET IN DIABETIC RETINOPATHY

    Abdelrahman, Ammar; Department of Biochemistry and Molecular Biology (Augusta University, 2020-12)
    Currently, treatments of diabetic retinopathy (DR) have limited therapeutic benefits and limited accessibility to the growing diabetic population at risk because of the high expenses and complicated procedures. Inflammation, endothelial dysfunction, and microvascular damage are common features of diabetic complications including DR. GPR109A is the metabolite sensing receptor of beta-hydroxybutyrate (BHB) the principal ketone body in humans. Our previous studies have shown the role of GPR109A expression in promoting anti-inflammatory response in retinal pigmented epithelial (RPE) cells and the relevance of the receptor in DR. Expression of the GPR109A in microvascular endothelial cells (ECs) has been reported recently. However, the relevance of GPR109A expression and activation to retinal EC functions are yet to be studied. Our goal in this study was to identify the role of GPR109A expression and activation in barrier and angiogenic functions of retinal ECs in context of diabetic retinopathy. We used electrical cell impedance sensing (ECIS) technology to evaluate barrier functions in primary human retinal endothelial cells (HRECs) which constitute the inner BRB. Knocking down GPR109A in HRECs with siRNA decreased the transendothelial electrical resistance (TEER) compared to scrambled siRNA. Treating HRECs with BHB increased their TEER and counteracted VEGF-induced barrier disruption through activation of GPR109A and increasing zonula occludens-1 (ZO-1) expression. Treatment of STZ-diabetic mice with exogenous BHB for one month protected against the pathologic albumin leakage induced by diabetes and improved the visual acuity of this animal model of diabetes. Using the mouse model of oxygen induced retinopathy (OIR), we showed that Gpr109a-/- mice had slower vascular recovery from pathologic angiogenesis compared to age matched wild type mice. Moreover, physiologic revascularization of vaso-oblitrated retinas was impaired by loss of GPR109a and associated with dysregulated inflammatory and angiogenic signaling. Collectively, these data point to a role for GPR109A in the regulation of barrier and angiogenic mechanisms in retinal ECs and, promote the receptor as a potential druggable target for impacting these mechanisms in microvascular retinal diseases such as DR.
  • A Molecular Basis of Chemoresistance in Bladder Cancer

    Lahorewala, Sarrah; Biochemistry and Cancer Biology (Augusta University, 2020-12)
    Background: In advanced bladder cancer (BC), development of resistance to the frontline chemotherapeutic drugs Gemcitabine and Cisplatin contributes to the poor prognosis of patients. Newly discovered chondroitinase, HYAL-4 V1 (V1), drives malignant transformation in BC. We evaluated V1’s role and the downstream molecules involved in the mechanistic regulation of chemoresistance in BC. Experimental Design: HYAL-4 expression was evaluated by RT-qPCR and IHC in metastatic muscle-invasive BC patients who received Gemcitabine plus Cisplatin chemotherapy. HYAL-4 wild-type and V1 were stably expressed or silenced in three BC and one normal urothelial cell line. Transfectants were analyzed for Gemcitabine and Cisplatin sensitivity, and for Gemcitabine influx and efflux to determine the mechanism of Gemcitabine resistance. The effect of cytidine deaminase (CDA) inhibition on Gemcitabine sensitivity was evaluated in vitro and in xenograft models. Results: HYAL-4 expression was an independent predictor of disease-specific mortality and treatment failure in our clinical cohort, and stratified patients into higher risk for both those outcomes. V1-expressing BC and normal urothelial cells were resistant to Gemcitabine due to the upregulation of cytidine deaminase (CDA) expression and activity, resulting in increased Gemcitabine metabolism and efflux; treating cells with tetrahydrouridine (THU), a CDA inhibitor, abrogated the chemotherapeutic resistance. Gemcitabine-resistant V1 cells demonstrated increased expression of V1’s substrate CD44 and phosphorylated STAT3. Si-RNA mediated CD44 knockdown and STAT3 inhibition both sensitized cells to Gemcitabine in vitro. In xenograft models, treatment with a combination of Gemcitabine and THU completely inhibited tumor growth. Conclusions: This project discovered V1 as a novel determinant of Gemcitabine resistance and potential predictor of treatment response in BC. V1 drives resistance to Gemcitabine through CD44-STAT3 mediated upregulation of CDA, and inhibiting this pathway sensitizes tumor cells to the therapy in preclinical models of BC.
  • Biomechanical behavior related to structure in normal and congenitally disordered elastic arteries

    Beall, Arthur C.; Department of Pharmacology and Toxicology (Augusta University, 1992-12)
  • The effects of retinoic acid-induced differentiation on neurotransmitter receptor content and signal transduction in a human neuroblastoma cell line

    Baumgartner, Melissa K.; Department of Pharmacology & Toxicology (Augusta University, 01/23/1993)
    The purpose of the present study was to establish the effects of retinoic acidindttced differentiation on muscarinic receptor populations and signal transduction pathways in the human neurroblastoma Sk-N-SH cells. The human neuroblastoma cell line Sk-N-SH was induced to differentiate by treatment with 1 uM retinoic acid for 7 days. Differentiation was characterized by profuse neurite outgrowth, a decrease in cell growth, and a 2~3 fold increase in the protein content of each cell. Muscarinic receptors were labelled-using [3H]N-methyl scopolamine. Muscarinic receptor density increased by approximately 36% after treatment for 7 days with retinoic acid (Bmax, control = 126 ± 13 fmol/mgprotein; Bmax, retinoic acid-treated= 170 ± 17 fmol/mg protein; p<0.05), corresponding to a 170% increase in receptor content per cell. The affinity of [3H]NMS for the receptors was somewhat lower in the differentiated cells (KD, control = 0.14 ± 0.04 nM; KD, retinoic acid-treated = 0.25 ± 0.0.4 nM; p<0.05). The guanine nucleotide sensitivity of agonist (carbamylcholine) binding to Sk-N-SH muscarinic receptors Was slightly decreased by differentiation. Reverse transcriptase/polymerase chain reaction (PCR) analysis using muscarinic receptor subtype specific primers revealed that the undifferentiatied Sk-N-SH cells transcribed mRNA for all 5 receptor subtypes; this pattern was not affected by differentiation. [3H]NMS displacement curves with subtype- selective receptor ligands (pirenzepine, m1; AFDX-116, m2; 4-DAMP, m3) indicated the predominant expression of m1 and m3 receptor subtypes, and differentiation did not affect the pharmacological profile of the expressed muscarinic receptor populations. Differentiation did not affect basal G protein GTPase activity. However, acetylcholine (100 uM) stimulation of G protein GTPase activity was decreased in differentiated cells (18 ± 1.8 pmol/min/mgprotein) compared to the undifferentiatied cells (23 ± 1 .0 pmol/ min/ mg protein) (p<0.05). Inhibition of acetylcholine--stimulated GTPase activity with selective muscarinic receptor antagonists indicated that the m3 antagonist (4-DAMP) was as effective as atropine in inhibiting activity by 80-100%. Selective m1 and m2 antagonists were less effective (30-40%) at inhibiting stimulated GTPase activity. There were no differences in inhibition of stimulated GTPase activity after differentiation. Immunoblots of control and retinoic acid-treated cells revealed no change in Goa, Gsa or Gp content after differentiation; however, 0.1% ethanol and retinoic acid-treated cells displayed a 30% decrease in expression of Gia3, and Gqa. Muscarine (0.1-100 uM) stimulated 45Ca influx into Sk-N-SH cells, and this uptake was inhibited by preincubation with atropine. The magnitude of the muscarinic receptor-mediated uptake was 50-60% lower in the differentiatied cells. Basal adenylate cyclase activity was depressed in the differentiated cells (2.5 pmol / min / mg protein) compared to the undifferentiated cells (8.4 pmol / min / mg protein) (p< 0.05). Forskolin (5 - 50 uM)-stimulated adenylate cyclase activity was not altered, however fractional stimulation was significantly (p<0.0001) increased in the differentiated cells. Differentiated cells displayed a slightly greater receptor-mediated inhibition of the adenylate cyclase activity by carbamylcholine (1 uM- 1 mM). It is demonstrated that in Sk-N-SH cells, retinoic acid-induced differentiation: 1) increases the size of the muscarinic receptor population (Bmax) while decreasing [3H]NMS binding affinity, 2) does not alter muscarinic receptor pharmacology, or the expression of. muscarinic receptor subtypes, 3) decreases muscarinic receptor-stimulated 45Ca flux 50-60% compared to undifferentiated cells, 4) depresses basal adenylate cyclase activity, increases fractional stimulation of forskolin-stimulated activity of adenylate cyclase, and may increase muscarinic receptor-mediated inhibition of adenylate cyclase activity, 5) does not alter basal G protein GTPase activity but depresses muscarinic receptor-stimulated high affinity GTPase activity suggesting muscarinic receptor-G protein coupling is altered, and 6) does not alter expression of Goa, Gsa and Gp content while Gia3 and Gqa are depressed in differentiated as well as in 0.1% e.thanol treated cells.

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