Name: Dissection of Receptor Subtypes that Control Smooth Muscle Contraction
Uploaded: 2022-07-11
Duration: 13 min 46 s
Description: Dissection of Receptor Subtypes that Control Smooth Muscle Contraction.

Audio Recording Jul 9, 2022 at 11:29:15 AM. Dissection of Receptor Subtypes that Control Smooth Muscle Contraction. 

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Dissection of Receptor Subtypes that Control Smooth Muscle Contraction

Group 2  Yu-Bian, Allie Casey, Yu-Wei Cheng, Aidan Cole, Alexis McCollum, Joshua Mercado,  Avani Parikh, and Breonna Runk

Audio Recording Jul 9, 2022 at 11:29:15 AM

Audio Recording Jul 9, 2022 at 12:00:25 PM. Background. 

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Novel drug was discovered and named  Drug X Similar function to Norepinephrine Discovered that  Drug X  targets multiple sites on Alpha Adrenergic receptors specifically Alpha-1   Adrenergic Receptor:  a  Gq  coupled receptor that promotes smooth muscle contraction and vasoconstriction Alpha-2 Adrenergic Receptor:   a Gi coupled receptor that promotes smooth muscle contraction with downstream signaling in coordination with Alpha-1

Sympathetic
nerve
Smooth
muscle
NE
(12
'CAMP
Contraction

Audio Recording Jul 9, 2022 at 12:00:25 PM

Audio Recording Jul 9, 2022 at 12:52:56. Background. 

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Treated uterine smooth muscle with  Drug X  and performed a Dose-Response Curve Observed a Hill slope more shallow than predicted for a single receptor binding site Cooperativity Positive - Substrate binding increases affinity for subsequent substrate binding Negative - Substrate binding decreases affinity for subsequent substrate binding Non-cooperative binding - Substrate binding does not affect subsequent substrate binding

Objective:  Determine whether our preliminary results are due to negative cooperativity or if Drug  X  is acting on multiple, independent receptors.

Experimental Design. Create Dose-Response curves using four different treatments: Positive Control: Cells and Drug X and antagonist solvent vehicle. (used to normalize response to 100%) Cells, Drug X and a competitive ɑ 1 antagonist (Prazosin, EC 50 9.05 nM). Cells, Drug X and a competitive ɑ 2 antagonist (Idazoxan, EC 50 6.37 nM). Negative Control: Cells and drug solvent vehicle (used to normalize response to 0%). 

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Create Dose-Response curves using four different treatments: Positive Control: Cells and Drug X and antagonist solvent vehicle. (used to normalize response to 100%) Cells, Drug X and a competitive ɑ 1  antagonist (Prazosin, EC 50  9.05 nM). Cells, Drug X and a competitive ɑ 2  antagonist (Idazoxan, EC 50  6.37 nM). Negative Control: Cells and drug solvent vehicle (used to normalize response to 0%)

Experimental Design (cont). Verify the antagonists with the same protocol using known antagonist of each receptor: Cells with Phenylepinephrine* and Prazosin or Idazoxan. Cells with Clonidine** and Prazosin or Idazoxan. *Known agonist of ɑ 1 receptors **Known agonist of ɑ 2 receptors. 

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Verify the antagonists with the same protocol using known antagonist of each receptor: Cells with Phenylepinephrine* and Prazosin or Idazoxan. Cells with Clonidine** and Prazosin or Idazoxan. *Known agonist of ɑ 1  receptors **Known agonist of ɑ 2  receptors

Audio Recording 10 Jul 2022 at 16:10:38. Analysis real experimental data with Prism. 

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Analysis real experimental data with Prism

Dose-response - Stimulation Equation: log(agonist) vs. response -- Variable slope

Many dose-response curves have a standard slope of 1.0. This model does not assume a standard slope but rather fits the Hill Slope from the data, and so is called a  Variable slope  model. It is also called a four-parameter dose-response curve, or four-parameter logistic curve, abbreviated 4PL. Model: Y=Bottom + (Top-Bottom)/(1+10^((LogEC50-X)*HillSlope)) EC50  is the concentration of agonist that gives a response half way between Bottom and Top. This is not the same as the response at Y=50

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Audio Recording Jul 10, 2022 at 7:44:39 PM. Validation of ? 1 and ? 2 known antagonists. 

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Validation of  ? 1  and  ? 2    known antagonists

Aim:  to ensure that known antagonists are selective in our model system

Audio Recording Jul 10, 2022 at 7:44:39 PM

Audio Recording Jul 10, 2022 at 8:09:08 PM. Selective inhibition of ? 1 and ? 2. 

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Selective inhibition of ? 1  reveals negative cooperativity

Audio Recording Jul 10, 2022 at 8:09:08 PM

Audio Recording Jul 10, 2022 at 20:41:47. Rigor and Reproducibility. 

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The Journal o
Physiology
Statistical Summary Document
Manuscript Title:
Authors:
Animal model used, if applicable:
I Underlying hypothesis:
Definitions of
Statistical summary table:
Experimental question
Multiple tests of the same question
are shown in different rows
[Provide one or two sentences]
[Define •n'. If definitions differ, please indicate which definition applies to which experimental question number.]
Finding/
conclusion
Mean value
location/variable
Standard
Deviation
(SD)
n
(value)
Exact P value
If P is considered significant
against a stated confidence
limit, authors may indicate
this in bold.The exact value
must be stated unless p is
less than in which
case < is permitted.
Figure/table Units
in which data
are presented
Data
comparisons
e.g. WTVS KO
Statistical
test
e.g. cortex vs
cerebellum or
(or other summary statistic)
Any other Comments
experiment
al factors
e.g. subjects' e.g. observation
age or sex

Statistical analysis will determine which conditions are statistically significant. It does not make inferences about the biological significance of the differences.  No statistical difference could mean the experiment is under-replicated.

Rigor and Reproducibility. Every experiment will have two technical replicates and 3 biological replicates Appropriate positive and negative controls Assess the quality of cell cultures (primary cells) and antagonist (Validation) Primary human uterine smooth muscle cells (HUtSMC) will be received from the American Type Culture Collection (ATCC)and tested regularly for mycoplasma contamination, cells maintained as recommended by ATCC Keep record of Source Catalog number Passage number of primary cells Organized and clear lab notebook Blinding and randomization to avoid potential bias Honesty is the best policy. 

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Every experiment will have two technical replicates and 3 biological replicates  Appropriate positive and negative controls Assess the quality of cell cultures (primary cells) and antagonist (Validation)  Primary human uterine smooth muscle cells (HUtSMC) will be received from the American Type Culture Collection (ATCC)and tested regularly for mycoplasma contamination, cells maintained as recommended by ATCC  Keep record of  Source  Catalog number Passage number of primary cells  Organized and clear lab notebook  Blinding and randomization to avoid potential bias  Honesty is the best policy

Acknowledgements & References. Background: Yu-Wei Cheng & Alexis McCollum Experimental Design: Aidan Cole & Joshua Mercado Analysis & Simulation: Allie Casey & Yu- Bian Rigor & Reproducibility: Avani Parikh & Breonna Runk References: Piascik , M. T. and D. M. Perez (2001). "Alpha1-adrenergic receptors: new insights and directions." J Pharmacol Exp Ther 298(2): 403-410. de Meyts , P. Cooperative properties of hormone receptors in cell membranes. Journal of Supramolecular Structure 4, 241–258 (1976). Casadevall, A. & Fang, F. C. Rigorous science: A how-to guide. mBio 7 , (2016).. 

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Background:  Yu-Wei Cheng & Alexis McCollum Experimental Design:  Aidan Cole & Joshua Mercado Analysis & Simulation:  Allie Casey & Yu- Bian Rigor & Reproducibility:   Avani  Parikh & Breonna  Runk References: Piascik , M. T. and D. M. Perez (2001). "Alpha1-adrenergic receptors: new insights and directions." J  Pharmacol  Exp  Ther  298(2): 403-410. de  Meyts , P. Cooperative properties of hormone receptors in cell membranes. Journal of Supramolecular Structure 4, 241–258 (1976). Casadevall, A. & Fang, F. C. Rigorous science: A how-to guide.  mBio   7 , (2016).

Dissection of Receptor Subtypes that Control Smooth Muscle Contraction

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