Biomaterial Database

Enzymatic isolation and characterization of single vascular smooth muscle cells from cremasteric arterioles. 1996

W F Jackson, and J M Huebner, and N J Rusch

Department of Biological Sciences, College of Arts and Sciences, Western Michigan University, Kalamazoo 49008, USA.

OBJECTIVE The goal of the present study was to develop a method to isolate enzymatically viable arteriolar muscle cells from single cremasteric arterioles, which retain the contractile and electrophysiological phenotype of the donor microvessels. METHODS Arterioles were hand-dissected from rat and hamster cremaster muscles and dissociated by incubation in papain and dithioerythritol for 35 min followed by incubation in collagenase, elastase, and soybean trypsin inhibitor for 10 to 25 min in solutions containing 100 microM Ca2+, 10 microM sodium nitroprusside, and 1 mg/ml albumin at 37 degrees C. RESULTS Populations of single smooth muscle cells enzymatically isolated from cremasteric arterioles showed elongated fusiform morphology and intact plasmalemmal membranes as indicated by retention of calcein, by exclusion of ethidium homodimer-1, and by high membrane resistances (11 +/- 0.8 G omega, n = 36 for rat cells; 8 +/- 0.6 G omega, n = 21 for hamster cells; p < 0.05). Muscle cells contracted in a concentration-dependent fashion in response to pipette application of norepinephrine (10 nM-100 microM). Cell shortening in response to 1 microM norepinephrine was inhibited by 10 microM phentolamine, 1 microM sodium nitroprusside, and 1 microM nifedipine or nominally Ca(2+)-free media. Resting membrane potential recorded in patch-clamped cells by perforated patch methods was -48 +/- 1 mV (n = 47) for rat cells and -44 +/- 2.8 mV (n = 14) for hamster cells (p > 0.05). Families of voltage-dependent K+ currents were observed during stepwise depolarizing pulses from -60 mV to more positive potentials. Blockers of voltage-gated and ATP-sensitive K+ channels (4-Aminopyridine [3 mM] and glibenclamide [1 microM], respectively) inhibited membrane K+ conductance, increased membrane resistance, and depolarized cells by 20 +/- 4 mV (n = 8) and 14 +/- 3 mV (n = 6), respectively. CONCLUSIONS The present method permits isolation of smooth muscle cells from a single cremasteric arteriole. These cells seem to retain the contractile phenotype, alpha-adrenergic signaling cascade, membrane potential, and K+ conductances described for the donor arteriole. Correlating the functional and electrophysiological properties of these smooth muscle cells to in situ and in vitro studies of their donor arterioles should provide a useful extension for understanding the physiology, pathophysiology, biophysics, and cell biology of the microcirculation in skeletal muscle.

UI	MeSH Term	Description	Entries
D008297	Male		Males
D008564	Membrane Potentials	The voltage differences across a membrane. For cellular membranes they are computed by subtracting the voltage measured outside the membrane from the voltage measured inside the membrane. They result from differences of inside versus outside concentration of potassium, sodium, chloride, and other ions across cells' or ORGANELLES membranes. For excitable cells, the resting membrane potentials range between -30 and -100 millivolts. Physical, chemical, or electrical stimuli can make a membrane potential more negative (hyperpolarization), or less negative (depolarization).	Resting Potentials,Transmembrane Potentials,Delta Psi,Resting Membrane Potential,Transmembrane Electrical Potential Difference,Transmembrane Potential Difference,Difference, Transmembrane Potential,Differences, Transmembrane Potential,Membrane Potential,Membrane Potential, Resting,Membrane Potentials, Resting,Potential Difference, Transmembrane,Potential Differences, Transmembrane,Potential, Membrane,Potential, Resting,Potential, Transmembrane,Potentials, Membrane,Potentials, Resting,Potentials, Transmembrane,Resting Membrane Potentials,Resting Potential,Transmembrane Potential,Transmembrane Potential Differences
D008647	Mesocricetus	A genus in the order Rodentia and family Cricetidae. One species, Mesocricetus auratus or golden hamster is widely used in biomedical research.	Hamsters, Golden,Hamsters, Golden Syrian,Hamsters, Syrian,Mesocricetus auratus,Syrian Golden Hamster,Syrian Hamster,Golden Hamster,Golden Hamster, Syrian,Golden Hamsters,Golden Syrian Hamsters,Hamster, Golden,Hamster, Syrian,Hamster, Syrian Golden,Syrian Hamsters
D009131	Muscle, Smooth, Vascular	The nonstriated involuntary muscle tissue of blood vessels.	Vascular Smooth Muscle,Muscle, Vascular Smooth,Muscles, Vascular Smooth,Smooth Muscle, Vascular,Smooth Muscles, Vascular,Vascular Smooth Muscles
D009543	Nifedipine	A potent vasodilator agent with calcium antagonistic action. It is a useful anti-anginal agent that also lowers blood pressure.	Adalat,BAY-a-1040,Bay-1040,Cordipin,Cordipine,Corinfar,Fenigidin,Korinfar,Nifangin,Nifedipine Monohydrochloride,Nifedipine-GTIS,Procardia,Procardia XL,Vascard,BAY a 1040,BAYa1040,Bay 1040,Bay1040,Monohydrochloride, Nifedipine,Nifedipine GTIS
D009599	Nitroprusside	A powerful vasodilator used in emergencies to lower blood pressure or to improve cardiac function. It is also an indicator for free sulfhydryl groups in proteins.	Nitroferricyanide,Sodium Nitroprusside,Cyanonitrosylferrate,Ketostix,Naniprus,Nipride,Nipruton,Nitriate,Nitropress,Nitroprussiat Fides,Nitroprusside, Disodium Salt,Nitroprusside, Disodium Salt, Dihydrate,Disodium Salt Nitroprusside,Nitroprusside, Sodium
D009638	Norepinephrine	Precursor of epinephrine that is secreted by the ADRENAL MEDULLA and is a widespread central and autonomic neurotransmitter. Norepinephrine is the principal transmitter of most postganglionic sympathetic fibers, and of the diffuse projection system in the brain that arises from the LOCUS CERULEUS. It is also found in plants and is used pharmacologically as a sympathomimetic.	Levarterenol,Levonorepinephrine,Noradrenaline,Arterenol,Levonor,Levophed,Levophed Bitartrate,Noradrenaline Bitartrate,Noradrénaline tartrate renaudin,Norepinephrin d-Tartrate (1:1),Norepinephrine Bitartrate,Norepinephrine Hydrochloride,Norepinephrine Hydrochloride, (+)-Isomer,Norepinephrine Hydrochloride, (+,-)-Isomer,Norepinephrine d-Tartrate (1:1),Norepinephrine l-Tartrate (1:1),Norepinephrine l-Tartrate (1:1), (+,-)-Isomer,Norepinephrine l-Tartrate (1:1), Monohydrate,Norepinephrine l-Tartrate (1:1), Monohydrate, (+)-Isomer,Norepinephrine l-Tartrate (1:2),Norepinephrine l-Tartrate, (+)-Isomer,Norepinephrine, (+)-Isomer,Norepinephrine, (+,-)-Isomer
D010646	Phentolamine	A nonselective alpha-adrenergic antagonist. It is used in the treatment of hypertension and hypertensive emergencies, pheochromocytoma, vasospasm of RAYNAUD DISEASE and frostbite, clonidine withdrawal syndrome, impotence, and peripheral vascular disease.	Fentolamin,Phentolamine Mesilate,Phentolamine Mesylate,Phentolamine Methanesulfonate,Phentolamine Mono-hydrochloride,Regitine,Regityn,Rogitine,Z-Max,Mesilate, Phentolamine,Mesylate, Phentolamine,Methanesulfonate, Phentolamine,Mono-hydrochloride, Phentolamine,Phentolamine Mono hydrochloride
D002118	Calcium	A basic element found in nearly all tissues. It is a member of the alkaline earth family of metals with the atomic symbol Ca, atomic number 20, and atomic weight 40. Calcium is the most abundant mineral in the body and combines with phosphorus to form calcium phosphate in the bones and teeth. It is essential for the normal functioning of nerves and muscles and plays a role in blood coagulation (as factor IV) and in many enzymatic processes.	Coagulation Factor IV,Factor IV,Blood Coagulation Factor IV,Calcium-40,Calcium 40,Factor IV, Coagulation
D002470	Cell Survival	The span of viability of a cell characterized by the capacity to perform certain functions such as metabolism, growth, reproduction, some form of responsiveness, and adaptability.	Cell Viability,Cell Viabilities,Survival, Cell,Viabilities, Cell,Viability, Cell