Impaired platelet-derived growth factor receptor expression and function in cultured lower esophageal sphincter circular smooth muscle cells from W/Wv mutant mice

We have previously demonstrated that lower esophageal sphincter (LES) circular smooth muscle (CSM) is functionally impaired in W/Wv mutant mice that lack interstitial cells of Cajal, and speculated that this could be due to altered smooth muscle differentiation. Platelet-derived growth factor (PDGF) is involved in the maturation and differentiation of smooth muscle. To determine whether PDGF expression and (or) function is altered in W/Wv mutant mice, PDGF-Rβ expression was measured using RT-PCR, qPCR, and immunocytochemistry, and Ca2+ imaging and perforated patch clamp recordings performed in isolated LES CSM cells. RT-PCR and immunocytochemistry showed significantly reduced PDGF-Rβ expression in the LES from mutant as opposed to wild-type mice. Quantitative comparison of CSM cell numbers in histological specimens revealed a significantly increased average cell size in the mutant tissue. The specific PDGF-Rβ ligand, PDGF-BB, caused a significant increase in intracellular Ca2+ in cells from the wild-type mice compared with the mutants. Using a ramp protocol, PDGF-BB caused a 2-fold increase in outward K+ currents in cells from the wild-type mice, whereas no significant increase was measured in the cells from the mutants. We conclude that the expression and function of PDGF-Rβ in LES CSM from W/Wv mice is impaired, providing further evidence that LES CSM is abnormal in W/Wv mutants.

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Neurotransmission in lower esophageal sphincter ofW/Wvmutant mice

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00266.2009 ◽

2010 ◽

Vol 298 (1) ◽

pp. G14-G24 ◽

Cited By ~ 33

Author(s):

Y. Zhang ◽

S. A. Carmichael ◽

X. Y. Wang ◽

J. D. Huizinga ◽

W. G. Paterson

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Lower Esophageal Sphincter ◽

Significant Proportion ◽

Muscle Cells ◽

Mutant Mice ◽

Resting Membrane Potential ◽

Wild Type ◽

Circular Smooth Muscle ◽

Esophageal Sphincter

To address the controversy surrounding the role of interstitial cells of Cajal (ICC) in nitrergic neurotransmission to gastrointestinal smooth muscle, circular smooth muscle from the lower esophageal sphincter (LES) of W/Wvwild-type and mutant (ICC-deficient) mice were studied by using intracellular and tension recordings in vitro. Resting membrane potential was more negative, and the spontaneous unitary potentials diminished in mutant mice. In wild-type mice, nerve stimulation induced a biphasic inhibitory junction potential (IJP) consisting of a fast initial IJP followed by a long-lasting slow IJP (LSIJP). The IJP was markedly impaired in a significant proportion of mutant mice, whereas in others it was normal. Pharmacological studies in the mice with markedly impaired IJPs revealed that cholinergic and purinergic components of the nerve-mediated responses appeared intact. In wild-type mice, caffeine hyperpolarized smooth muscle cells, inhibited the initial fast IJP, and completely abolished the LSIJP. In mutant mice, caffeine depolarized smooth muscle cells and abolished the impaired LSIJP but did not affect the initial fast IJP. Immunohistochemical staining for c-Kit confirmed deficiency of ICC in mutant mice with a normal nitrergic IJP. Rings of LES circular smooth muscle from W/Wvmutant mice generated significantly less spontaneous tone than controls. When tone was restored with carbachol, normal nitrergic LES relaxation was recorded. These data suggest that 1) there is significant variability in the generation of nitrergic neurotransmission in the LES of W/Wvmutant mice, whereas purinergic and cholinergic neurotransmission are intact; 2) the altered nitrergic responses appear to be associated with abnormal Ca2+-dependent signaling initiated by spontaneous Ca2+release from sarcoplasmic reticulum in smooth muscle cells; and 3) c-Kit-positive ICC are not essential for nitrergic neurotransmission in mouse LES smooth muscle.

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Evidence for altered circular smooth muscle cell function in lower esophageal sphincter ofW/Wvmutant mice

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00020.2011 ◽

2011 ◽

Vol 301 (6) ◽

pp. G1059-G1065 ◽

Cited By ~ 4

Author(s):

Francisco Bautista-Cruz ◽

William G. Paterson

Keyword(s):

Smooth Muscle ◽

Membrane Potential ◽

Lower Esophageal Sphincter ◽

Mutant Mice ◽

Resting Membrane Potential ◽

Circular Smooth Muscle ◽

Significant Difference ◽

Esophageal Sphincter ◽

And Control ◽

In Cells

Nitrergic neurotransmission to gut smooth muscle is impaired in W/Wvmutant mice, which lack intramuscular interstitial cells of Cajal (ICC-IM). In addition, these mice have been reported to have smaller amplitude unitary potentials (UPs) and a more negative resting membrane potential (RMP) than control mice. These abnormalities have been attributed to absence of ICC-IM, but it remains possible that they are due to alterations at the level of the smooth muscle itself. Amphotericin-B-perforated patch-clamp recordings and Ca2+imaging (fura 2) were compared between freshly isolated single circular smooth muscle cells (CSM) from W/Wvmutant and control mice lower esophageal sphincter (LES). There was no significant difference in seal resistance, capacitance, or input resistance in response to applied electrotonic current pulses between CSM cells from W/Wvmutants and controls. Compared with control mice, RMP was more negative and UPs significantly smaller in CSM cells from mutant mice LES. Administration of caffeine induced an inward current in cells from both mutant and control mice, but the current density was significantly larger in cells from W/Wvmutants. Membrane potential hyperpolarization induced by sodium nitroprusside was larger in cells from control mice vs. W/Wvmutants. In addition, intracellular Ca2+transients induced by caffeine were significantly increased in cells from mutants. These findings indicate that LES CSM is abnormal in W/Wvmutant mice. Thus some physiological functions attributed to ICC-IM based on experiments in smooth muscle of ICC deficient mice may need to be reconsidered.

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