The authors would like to acknowledge the National Research Council of Thailand, the Center of Excellence for Innovation in Chemistry (PERCH-CIC), and the International Research Network (IRN61W0005) for providing financial support, and the Department of Physiology Faculty of Medical Science, Naresuan University, for research facility support.

The experiments performed in this study were approved by the Ethics Committee of Naresuan University Animal Care and Use Committee (NUACUC), protocol number NU-AE620921, for the care and use of animals for scientific purposes.
1. Composition of physiological solutions
<ol>
	<li>Formulate Krebs solution by dissolving chemicals in distilled water to achieve the final concentrations as follows: 122 mM NaCl, 10 mM HEPES, 5 mM KCl, 0.5 mM KH2PO4, 0.5 mM NaHP...

<ol>
	<li>Lyle, M. A., Davis, J. P., Brozovich, F. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+pulmonary+vascular+smooth+muscle+contractility+in+pulmonary+arterial+hypertension:+Implications+for+therapy.">Regulation of pulmonary vascular smooth muscle contractility in pulmonary arterial hypertension: Implications for therapy.</a> Frontiers in Physiology. 8, 614 (2017).</li><li>Cyr, A. R., Huckaby, L. V., Shiva, S. S., Zuckerbraun, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+and+endothelial+dysfunction.">Nitric oxide and endothelial dysfunction.</a> Critical Care Clinics. 36 (2), 307-321 (2020).</li><li>Ruan, K. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advance+in+understanding+the+biosynthesis+of+prostacyclin+and+thromboxane+A2+in+the+endoplasmic+reticulum+membrane+via+the+cyclo-oxygenase+pathway.">Advance in understanding the biosynthesis of prostacyclin and thromboxane A2 in the endoplasmic reticulum membrane via the cyclo-oxygenase pathway.</a> Mini Reviews in Medicinal Chemistry. 4 (6), 639-647 (2004).</li><li>Del Pozo, R., Hernandez Gonzalez, I., Escribano-Subias, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prostacyclin+pathway+in+pulmonary+arterial+hypertension:+A+clinical+review.">The prostacyclin pathway in pulmonary arterial hypertension: A clinical review.</a> Expert Review of Respiratory Medicine. 11 (6), 491-503 (2017).</li><li>Morgado, M., Cairr&#227;o, E., Santos-Silva, A. J., Verde, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+nucleotide-dependent+relaxation+pathways+in+vascular+smooth+muscle.">Cyclic nucleotide-dependent relaxation pathways in vascular smooth muscle.</a> Cellular and Molecular Life Sciences. 69 (2), 247-266 (2012).</li><li>Schmidt, K., de Wit, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelium-derived+hyperpolarizing+factor+and+myoendothelial+coupling:+The+in+vivo+perspective.">Endothelium-derived hyperpolarizing factor and myoendothelial coupling: The in vivo perspective.</a> Frontiers in Physiology. 11, (2020).</li><li>Fan, G., Cui, Y., Gollasch, M., Kassmann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elementary+calcium+signaling+in+arterial+smooth+muscle.">Elementary calcium signaling in arterial smooth muscle.</a> Channels. 13 (1), 505-519 (2019).</li><li>Wisutthathum, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extract+of+Aquilaria+crassna+leaves+and+mangiferin+are+vasodilators+while+showing+no+cytotoxicity.">Extract of Aquilaria crassna leaves and mangiferin are vasodilators while showing no cytotoxicity.</a> Journal of Traditional and Complementary Medicine. 9 (4), 237-242 (2019).</li><li>Kamkaew, N., Paracha, T. U., Ingkaninan, K., Waranuch, N., Chootip, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasodilatory+effects+and+mechanisms+of+action+of+Bacopa+monnieri+active+compounds+on+rat+mesenteric+arteries.">Vasodilatory effects and mechanisms of action of Bacopa monnieri active compounds on rat mesenteric arteries.</a> Molecules. 24 (12), 2243 (2019).</li><li>Chootip, K., Kennedy, C., Gurney, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+P2+receptors+mediating+contraction+of+the+rat+isolated+pulmonary+vasculature.">Characterization of P2 receptors mediating contraction of the rat isolated pulmonary vasculature.</a> British Journal of Pharmacology. 131, 167 (2000).</li><li>Paracha, T. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidation+of+vasodilation+response+and+structure+activity+relationships+of+N2,+N4-disubstituted+quinazoline+2,+4-diamines+in+a+rat+pulmonary+artery+model.">Elucidation of vasodilation response and structure activity relationships of N2, N4-disubstituted quinazoline 2, 4-diamines in a rat pulmonary artery model.</a> Molecules. 24 (2), 281 (2019).</li><li>Chootip, K., Gurney, A. M., Kennedy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+P2Y+receptors+couple+to+calcium-dependent,+chloride+channels+in+smooth+muscle+cells+of+the+rat+pulmonary+artery.">Multiple P2Y receptors couple to calcium-dependent, chloride channels in smooth muscle cells of the rat pulmonary artery.</a> Respiratory Research. 6 (1), 1-10 (2005).</li><li>Wisutthathum, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eulophia+macrobulbon+extract+relaxes+rat+isolated+pulmonary+artery+and+protects+against+monocrotaline-induced+pulmonary+arterial+hypertension.">Eulophia macrobulbon extract relaxes rat isolated pulmonary artery and protects against monocrotaline-induced pulmonary arterial hypertension.</a> Phytomedicine. 50, 157-165 (2018).</li><li>Kruangtip, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curcumin+analogues+inhibit+phosphodiesterase-5+and+dilate+rat+pulmonary+arteries.">Curcumin analogues inhibit phosphodiesterase-5 and dilate rat pulmonary arteries.</a> Journal of Pharmacy and Pharmacology. 67 (1), 87-95 (2015).</li></ol>

In this manuscript, we describe the technique for the isolation of rat IPA and VSMCs. Several experimental protocols have been employed to investigate the vascular response of IPA in vitro, which can be used to characterize the pharmacological effect and mechanistic basis of IPA vasorelaxation induced by plant extract.
Regarding the endothelium-dependent vasodilator action of the plant extract, various blockers such as L-NAME (eNOS), indomethacin (COX), and apamin+charybdotoxin (EDHF)...

The pulmonary vasculature is a low-pressure vascular system in which the main function is to deliver deoxygenated blood to the gas exchanging area of the lungs. The pulmonary arteries in the lungs are arranged in branches parallel to the bronchial tree, ultimately forming an extensive network of capillaries that is continuous over several alveoli and, finally, coming together into venules and veins. The vascular tone of the pulmonary artery is controlled by several factors, involving the interaction between the endothelium and vascular smooth muscle cells (VSMCs)1.
In this study, we focus on the endothelium-dependent and -independent vasorelaxation of the intrapulmonary artery (IPA). With regard to the endothelium-dependent vasorelaxation, various mechanisms occurring on the surface of endothelial cells could increase intracellular Ca2+ concentration (e.g., acetylcholine [ACh] binds with muscarinic receptor [M3]), leading to the formation of nitric oxide (NO), prostacyclin (PGl2) and endothelium-derived hyperpolarizing factor (EDHF) (Figure 1). NO is the main endothelium-derived relaxing factor synthesized from L-arginine by endothelial nitric oxide synthase (eNOS)2, which then dissociates out of the endothelial cells to VSMCs (Figure 1) and stimulates the soluble guanylyl cyclase (sGC) enzyme; this enzyme changes guanosine triphosphate (GTP) into cyclic guanosine monophosphate (cGMP), which activates protein kinase G (PKG) and reduces cytosolic Ca2+ levels, thus causing vasorelaxation (Figure 1). PGl2 is synthesized by endothelial cells via the cyclo-oxygenase (COX) pathway3,4. It binds with the prostacyclin receptor (IP) on VSMCs and stimulates the adenylyl cyclase (AC) enzyme, which then converts adenosine triphosphate (ATP) into cyclic adenosine monophosphate (cAMP) (Figure 1)3,4. cAMP activates protein kinase A (PKA), reducing cytosolic Ca2+ levels and causing vasorelaxation5 (Figure 1). The EDHF pathway also participates in endothelium-dependent vasorelaxation via various endothelial mediators and electrical events. The activation of the EDHF pathway leads to the hyperpolarization of VSMCs, thus closing voltage-operated Ca2+ channels (VOCCs), reducing intracellular Ca2+ levels, and inducing vasorelaxation6. The endothelium-independent vasorelaxation occurs directly on VSMCs via several mechanisms, such as the reduction of intracellular Ca2+ level, the inhibition of myosin light chain kinase (MLCK), the activation of myosin light chain phosphatase (MLCP), and the reduction of Ca2+ sensitivity to the contractile machinery of VSMCs. In this study, we focus on the vasorelaxation caused by the opening of various K+ channels, the blockade of VOCCs, and the inhibition of Ca2+ release from the sarcoplasmic reticulum7, which leads to the reduction of intracellular Ca2+ levels, thus decreasing VSMC myosin light chain phosphorylation and myosin-actin binding or cross-bridge formation, respectively, ultimately resulting in vasorelaxation.
The technique for evaluating vasoconstriction and vasorelaxation measurements in isolated IPA is well established for rodents, but the data varied depending on the experimental protocols. The present study describes the method used to evaluate the vascular reactivities of rat IPA preparations in vitro, which were made in the absence of external factors modulating vascular response in vivo, such as nerve signals, hormones, cytokines, blood pressure, etc.
We employed several experimental protocols using the plant extract as an example for studying the vascular reactivities of IPA. Various blockers (Figure 1) were utilized to identify the mechanisms of endothelium-dependent and -independent vasorelaxation induced by the plant extract. Nevertheless, the same protocols can be adapted to evaluate the vascular responses of IPA to any drugs, extracts or phytochemicals used for the treatment of various pulmonary pathologies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,4-dithiothreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td>D0632 
			CAS NO. 348-12-3</td>
			<td></td>
		</tr>
		<tr>
			<td>4-aminopyridine (4-AP)</td>
			<td>Aldrich Chemical</td>
			<td>A78403 
			CAS NO. 504-24-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetylcholine</td>
			<td>Sigma-Aldrich</td>
			<td>A6625 
			CAS NO. 60-31-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Apamin</td>
			<td>Sigma-Aldrich</td>
			<td>A9459 
			CAS NO. 24345-16-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A2153 
			CAS NO. 9048-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium choride</td>
			<td>Ajax Finechem</td>
			<td>AJA960 
			CAS NO. 1707055184</td>
			<td></td>
		</tr>
		<tr>
			<td>Charybdotoxin</td>
			<td>Sigma-Aldrich</td>
			<td>C7802 
			CAS NO. 95751-30-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type 1A</td>
			<td>Sigma-Aldrich</td>
			<td>C9891 
			CAS NO. 9001-12-1</td>
			<td>From Clostridium histolyticum</td>
		</tr>
		<tr>
			<td>D(+)-Glucose monohydrate</td>
			<td>Millipore Corporation</td>
			<td>K50876942 924 
			CAS NO. 14431-43-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D4540 
			CAS NO. 67-68-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis (2-aminoethylether)-N,N,N&#8217;,N&#8217;-tetraacetic acid (EGTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E3889 
			CAS NO. 67-42-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E9884 
			CAS NO. 60-00-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps 11 cm.</td>
			<td>Rustless Dumoxel</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps 14 cm.</td>
			<td>Rustless Dumoxel</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Glibenclamide</td>
			<td>Sigma-Aldrich</td>
			<td>G6039 
			CAS NO. 16673-34-0</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism program</td>
			<td>Software version 5.0 (San Diego, CA, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375 
			CAS NO. 7365-45-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Iberiotoxin</td>
			<td>Sigma-Aldrich</td>
			<td>I5904 
			CAS NO. 1002546960</td>
			<td>recombinant from Mesobuthus tamulus</td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Sigma-Aldrich</td>
			<td>I7378 
			CAS NO. 53-86-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Labchart Program</td>
			<td>Software version 7.0 (A.D. Instrument, Castle Hill, Australia).</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Ajax Finechem</td>
			<td>296 
			CAS NO. 1506254995</td>
			<td></td>
		</tr>
		<tr>
			<td>Male Wistar rats</td>
			<td>Nomura Siam International Co. Ltd., Bangkok, Thailand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NG-nitro-L-arginine methyl ester (L-NAME)</td>
			<td>Sigma-Aldrich</td>
			<td>N5751 
			CAS NO. 51298-62-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicardipine</td>
			<td>Sigma-Aldrich</td>
			<td>N7510 
			CAS NO. 54527-84-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Organ bath 15 mL.</td>
			<td>-</td>
			<td>-</td>
			<td>Specific order by the researchers</td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Sigma-Aldrich</td>
			<td>P4762 
			CAS NO. 9001-73-4</td>
			<td>FromPapaya Latex</td>
		</tr>
		<tr>
			<td>Phenal red</td>
			<td>Sigma-Aldrich</td>
			<td>P5530 
			CAS NO. 34487-61-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylephrine</td>
			<td>Sigma-Aldrich</td>
			<td>P6126 
			CAS NO. 61-76-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Kemaus</td>
			<td>KA383 
			CAS NO. 7447-40-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogenphosphate</td>
			<td>Aldrich Chemical</td>
			<td>EC231-913-4 
			CAS NO. 7778-77-0</td>
			<td></td>
		</tr>
		<tr>
			<td>S+A2:E36odium chloride</td>
			<td>Kemaus</td>
			<td>KA465 
			CAS NO. 7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors 11 cm.</td>
			<td>Spall Stainless</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors 14 cm.</td>
			<td>Spall Stainless</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Ajax Finechem</td>
			<td>475 
			CAS NO. 912466</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogenphosphate</td>
			<td>Aldrich Chemical</td>
			<td>33,198-8 
			CAS NO. 7558-80-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Ajax Finechem</td>
			<td>482 
			CAS NO. 1506196602</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium thiopental</td>
			<td>Anesthal</td>
			<td>JPN3010002 
			CAS NO. 1C 314/47</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich</td>
			<td>T0625 
			CAS NO. 107-35-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Waterbath WBU 45</td>
			<td>Memmert</td>
			<td>2766 
			CAS NO. -</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of intrapulmonary artery and smooth muscle cells to investigate vascular responses

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of vasoconstriction and vasorelaxation. After isolating the IPA and VSMCs, the characteristics of vascular responses in physiological and pathological conditions can be assessed in the absence of extrinsic factors such as nerve signals, hormones, cytokines, etc. Thus, the IPA and VSMCs serve as excellent models for studying vascular physiology/pathophysiology, along with various experimental investigations, such as modulation by pharmacological agents, patch-clamp electrophysiological analysis, calcium imaging, etc. Here, we have used a technique for isolating the IPA to investigate vascular responses in an organ bath setup. IPA segments were mounted on the organ bath chamber via intraluminal wires and stimulated by various pharmacological agents. The changes in IPA vascular tone (i.e., vasoconstriction and vasorelaxation), were recorded using an isometric force transducer and physiological data analysis software program. We implemented several experimental protocols, which can be adapted to investigate the mechanisms of vasorelaxation/vasoconstriction for studying the pharmacological activities of phytochemical or synthetic drugs. The protocols can also be used to evaluate drugs&#39; roles in modulating various diseases, including pulmonary arterial hypertension. The IPA model allows us to investigate the concentration-response curve, which is crucial in assessing drugs&#39; pharmacodynamic parameters.

The protocol in the present study has been developed to determine the optimal experimental conditions for measuring physiological phenomena observed in the vascular responses of isolated IPA preparations. The pilot experiments were performed to describe the potential outcomes that aid the understanding of the vascular effects and mechanistic basis of the vasorelaxant action of the plant extract, as follows.
Vasorelaxant effect of the plant extract 
As shown in <strong cla...

Watch this Scientific Journal Video about Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses at JoVE.com

Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses

Vascular responses of arterial pulmonary circulation can be explored using intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs). The present study describes the isolation of IPA in detail and the protocols used for investigating vasorelaxation in response to physiological stimuli.

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of ...

This study describes isolating rat intrapulmonary artery and vascular smooth muscle cells. Several experimental protocols were employed, such as using the organ bath technique to investigate vascular response of the intrapulmonary artery. Intrapulmonary arteries and vascular smooth muscle cells are excellent models to study vascular physiology and pathophysiology.Adaptations of this technique could evaluate vascular responses of any drugs, extracts or phytochemicals. Mounting the isolated intrapulmonary arterial ring in an organ bath enables the use of isolated intrapulmonary arteries to evaluate a drug's role in modulating various diseases, including pulmonary hypertension. The experimental protocols are technically feasible, but the crucial steps are complicated and essential for success, so having a visual demonstration makes it easier to understand and follow.Demonstrating the procedure will be Kittiwoot To-on, a master's degree student in physiology from the Cardiovascular Research Unit Laboratory, Department of Physiology, Faculty of Medical Science, Naresuan University. Begin by slicing a single lobe of the lung with 11-centimeter scissors and placing it on a nine-centimeter Petri dish with the medial side, or root of the lung, facing upward. Observe and identify the alignment of the vein, bronchia, and artery from top to bottom.Cut open the bronchus longitudinally with scissors. Then use the 11-centimeter forceps to grab the tip of the bronchus. Gently dissect and remove the bronchus and veins out of the lung.Use the forceps to grab the tip of the intrapulmonary artery and carefully dissect it out of the lung tissue with scissors. Keep the isolated intrapulmonary artery in cold Krebs solution until the organ bath assembly is set up. After isolating the intrapulmonary artery, cut open the main branch of the intrapulmonary artery longitudinally with the 11-centimeter scissors and cut into two-millimeter strips.Immerse the intrapulmonary artery strips in DM, then incubate the strips for one hour at four degrees Celsius in DM containing one milligram per milliliter papain, 0.04%BSA and 0.4 millimolar DTT. Then incubate at 37 degrees Celsius for 15 minutes. Add one milligram per milliliter of collagenase Type IA in DM, and again incubate at 37 degrees Celsius for five minutes.Transfer the tissues into fresh DM and disperse by gentle trituration using a glass Pasteur pipette. Keep triturating until isolated vascular smooth muscle cells become visible in the bathing solution under the microscope. Isolate intrapulmonary artery as demonstrated earlier and cut the main branch of the intrapulmonary artery into rings of approximately two millimeters in length.Affix the intrapulmonary artery rings in organ bath chambers by threading them onto two 40-micrometer-diameter stainless steel wires. Attach stainless steel wires mounted with intrapulmonary artery rings to the isometric force transducers connected to the data acquisition device and the computer system installed with the suitable physiological software for data recording and analysis. Then gently raise the tension of the intrapulmonary artery ring to one gram.Allow the vessel segments to equilibrate for about 45 minutes at a resting tension of one gram. During the equilibration period, ensure that the Krebs solution is regularly changed every 15 minutes. After equilibration, test the viability of the vessels by measuring their vasoconstriction to high extracellular potassium solution.Assess the presence or absence of endothelium by computing the relaxation response to acetylcholine in rings precontracted with PE.Mechanically remove the endothelium by gently rubbing the inside of the vessel with a small wire to induce denudation. Then equilibrate the arterial rings for 30 minutes before the start of the test experiments. Investigate the relaxant effect of the plant extract by precontracting intrapulmonary artery rings with PE.Then carefully add 0.1 to 1000 micrograms per milliliters of the plant extract cumulatively to endothelium intact rings and endothelium-denuded rings to induce vasorelaxation and acquire a concentration-dependent response curve.Ensure that the effective DMSO used as a solvent is also evaluated similarly to serve as a negative control. Evaluate the vasorelaxant mechanism of action of the plant extract as mentioned in the text manuscript. Then after the contractions to PE stabilize, add cumulative concentrations of the plant extract.Present the effects of plant extract as percentage relaxation of the intrapulmonary artery rings in the presence of inhibitors compared to the response of the intrapulmonary artery rings without inhibitors and construct the concentration response curve. In endothelium intact intrapulmonary artery, the plant extract elicited a concentration-dependent response of vasorelaxation. Eradication of endothelium profoundly reduced the vasorelaxation induced by the plant extract as reflected by the increase in the EC50 by 2.2 fold, inhibition of eNOS and EDHF evidently decreased the vasorelaxant response to the plant extract.This shifted the concentration response curve to the right and increased the EC50 without altering the E max values. On the contrary, Indomethacin showed no effect on the vasorelaxant response to the plant extract. In the endothelium-denuded intrapulmonary artery rings, calcium-activated potassium channel blockers decreased the vasorelaxant response to the plant extract.While blockers of voltage-gated or ATP-sensitive potassium channels showed no deviation. Preincubation with the plant extract inhibited the calcium-chloride-induced contraction. The endothelium-denuded intrapulmonary artery rings preincubated with calcium-free Krebs solution and PE rendered a transient contraction.In comparison to the vehicle, the plant extracts significantly reduced the contraction induced by PE.During the procedure, one must clearly identify vein, bronchia, and artery early on. Cutting or damaging the artery will reduce its length and affect the vascular responses. This technique can be adapted to investigate compounds that might serve as prototypes for treatments of pulmonary vascular diseases such as pulmonary arterial hypertension.

isolation-intrapulmonary-artery-smooth-muscle-cells-to-investigate

Research

JoVE Journal

Medicine

2.8K vistas.  Naresuan University. Este estudio describe el aislamiento de la arteria intrapulmonar de rata y las células del músculo liso vascular. Se emplearon varios protocolos experimentales, como el uso de la técnica de baño de órganos para investigar la respuesta vascular de la arteria intrapulmonar. Las arterias intrapulmonares y las células del músculo liso vascular son excelentes modelos para estudiar la fisiología vascular y la fisiopatología.Las adaptaciones de esta técnica podrían evaluar las resp...