Tu2001 Development of Myogenic IAS Reconstructs From Human Internal Anal Sphincter (IAS) Smooth Muscle Cells (Smcs) With Functional and Molecular Properties Similar to Intact Human IAS

2012 ◽  
Vol 142 (5) ◽  
pp. S-898
Author(s):  
Satish C. Rattan ◽  
Jagmohan Singh
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Anal Sphincter ◽  
Internal Anal Sphincter ◽  
Muscle Cells ◽  
Molecular Properties

scholarly journals Rhythmic calcium transients in smooth muscle cells of the mouse internal anal sphincter

2019 ◽  
Vol 32 (3) ◽  
Author(s):  
Caroline A. Cobine ◽  
Karen I. Hannigan ◽  
Megan McMahon ◽  
Emer P. Ni Bhraonain ◽  
Salah A. Baker ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Anal Sphincter ◽  
Internal Anal Sphincter ◽  
Muscle Cells ◽  
Calcium Transients

Mechanism of internal anal sphincter smooth muscle relaxation by phorbol 12,13-dibutyrate

2001 ◽  
Vol 280 (6) ◽  
pp. G1341-G1350 ◽  
Author(s):  
Sushanta Chakder ◽  
D. N. K. Sarma ◽  
Satish Rattan
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Anal Sphincter ◽  
Internal Anal Sphincter ◽  
Inhibitory Action ◽  
Muscle Tone ◽  
Muscle Relaxation ◽  
Muscle Cells ◽  
Smooth Muscle Relaxation ◽  
Smooth Muscle Tone

We investigated the mechanism of the inhibitory action of phorbol 12,13-dibutyrate (PDBu), one of the typical protein kinase C (PKC) activators, in in vitro smooth muscle strips and in isolated smooth muscle cells of the opossum internal anal sphincter (IAS). The inhibitory action of PDBu on IAS smooth muscle (observed in the presence of guanethidine + atropine) was partly attenuated by tetrodotoxin, suggesting that a part of the inhibitory action of PDBu is via the nonadrenergic, noncholinergic neurons. A major part of the action of PDBu in IAS smooth muscle was, however, via its direct action at the smooth muscle cells, accompanied by a decrease in free intracellular Ca2+ concentration ([Ca2+]i) and inhibition of PKC translocation. PDBu-induced IAS smooth muscle relaxation was unaffected by agents that block Ca2+ mobilization and Na+-K+-ATPase. The PDBu-induced fall in basal IAS smooth muscle tone and [Ca2+]i resembled that induced by the Ca2+ channel blocker nifedipine and were reversed specifically by the Ca2+ channel activator BAY K 8644. We speculate that a major component of the relaxant action of PDBu in IAS smooth muscle is caused by the inhibition of Ca2+ influx and of PKC translocation to the membrane. The specific role of PKC downregulation and other factors in the phorbol ester-mediated fall in basal IAS smooth muscle tone remain to be determined.

scholarly journals Successful implantation of physiologically functional bioengineered mouse internal anal sphincter

2010 ◽  
Vol 299 (2) ◽  
pp. G430-G439 ◽  
Author(s):  
Shreya Raghavan ◽  
Eiichi A. Miyasaka ◽  
Mohamed Hashish ◽  
Sita Somara ◽  
Robert R. Gilmont ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Anal Sphincter ◽  
Intracellular Signaling ◽  
Internal Anal Sphincter ◽  
Muscle Cells ◽  
Nitric Oxide Donor ◽  
Proof Of Concept ◽  
Smooth Muscle Tissue ◽  
Basal Tone

We have previously developed bioengineered three-dimensional internal anal sphincter (IAS) rings from circular smooth muscle cells isolated from rabbit and human IAS. We provide proof of concept that bioengineered mouse IAS rings are neovascularized upon implantation into mice of the same strain and maintain concentric smooth muscle alignment, phenotype, and IAS functionality. Rings were bioengineered by using smooth muscle cells from the IAS of C57BL/6J mice. Bioengineered mouse IAS rings were implanted subcutaneously on the dorsum of C57BL/6J mice along with a microosmotic pump delivering fibroblast growth factor-2. The mice remained healthy during the period of implantation, showing no external signs of rejection. Mice were killed 28 days postsurgery and implanted IAS rings were harvested. IAS rings showed muscle attachment, neovascularization, healthy color, and no external signs of infection or inflammation. Assessment of force generation on harvested IAS rings showed the following: 1) spontaneous basal tone was generated in the absence of external stimulation; 2) basal tone was relaxed by vasoactive intestinal peptide, nitric oxide donor, and nifedipine; 3) acetylcholine and phorbol dibutyrate elicited rapid-rising, dose-dependent, sustained contractions repeatedly over 30 min without signs of muscle fatigue; and 4) magnitudes of potassium chloride-induced contractions were 100% of peak maximal agonist-induced contractions. Our preliminary results confirm the proof of concept that bioengineered rings are neovascularized upon implantation. Harvested rings maintain smooth muscle alignment and phenotype. Our physiological studies confirm that implanted rings maintain 1) overall IAS physiology and develop basal tone, 2) integrity of membrane ionic characteristics, and 3) integrity of membrane associated intracellular signaling transduction pathways for contraction and relaxation by responding to cholinergic, nitrergic, and VIP-ergic stimulation. IAS smooth muscle tissue could thus be bioengineered for the purpose of implantation to serve as a potential graft therapy for dysfunctional internal anal sphincter in fecal incontinence.

171 Physiology of Intrinsically Innervated Bioengineered Construct From Human Internal Anal Sphincter Smooth Muscle Cells

2010 ◽  
Vol 138 (5) ◽  
pp. S-31
Author(s):  
Shreya Raghavan ◽  
Robert R. Gilmont ◽  
Sita Somara ◽  
Shanthi Srinivasan ◽  
Khalil Bitar
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Anal Sphincter ◽  
Internal Anal Sphincter ◽  
Muscle Cells

Evidence for VIP-induced increase in NO production in myenteric neurons of opossum internal anal sphincter

1996 ◽  
Vol 270 (3) ◽  
pp. G492-G497 ◽  
Author(s):  
S. Chakder ◽  
S. Rattan
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Anal Sphincter ◽  
Internal Anal Sphincter ◽  
Muscle Cells ◽  
No Production ◽  
Myenteric Neurons ◽  
No Release ◽  
Nos Inhibitor ◽  
Myenteric Ganglia

A significant interaction between vasoactive intestinal polypeptide (VIP) and nitric oxide (NO) has been reported in neurotransmission of the gastrointestinal tract, including the internal anal sphincter (IAS). The exact site of this NO release from the IAS in response to VIP is not known. Studies were carried out to determine the site of this VIP-induced NO release in opossum IAS. NO synthase (NOS) activity was quantitated by determining L--3H-citrulline production from L[3H]arginine in isolated myenteric ganglia and smooth muscle cells of the IAS. L-[3H]citrulline production was determined before and after treatment with either the ganglionic stimulant 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), VIP, or peptide histidine isoleucine (PHI) in the absence and presence of the neurotoxin tetrodotoxin and the NOS inhibitor NG-nitro-L-arginine (L-NNA). Smooth muscle cells and ganglia were preloaded with L-[3H]arginine for 5 min and treated with VIP for 1 and 5 min. DMPP and VIP caused a significant increase in L-[3H]citrulline formation in myenteric ganglia at both time periods, whereas in smooth muscle cells there was a moderate but significant increase in L-[3H]citrulline production only at 5 min of VIP treatment. VIP-induced relaxation of isolated smooth muscle cells of the IAS was not affected by L-NNA. The increase in NOS activity of myenteric ganglia by DMPP and VIP was sensitive to neurotoxin and the NOS inhibitor. The data suggest that the increase in NO production in response to VIP in the IAS occurs mainly from the myenteric neurons, with some contribution from the smooth muscle cells.

scholarly journals Immunocytochemical evidence for PDBu-induced activation of RhoA/ROCK in human internal anal sphincter smooth muscle cells

2011 ◽  
Vol 301 (2) ◽  
pp. G317-G325 ◽  
Author(s):  
Jagmohan Singh ◽  
Pinckney J. Maxwell ◽  
Satish Rattan
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Anal Sphincter ◽  
Internal Anal Sphincter ◽  
Muscle Cells ◽  
M Cell ◽  
Rock Inhibitor ◽  
Calphostin C ◽  
Relative Contribution ◽  
C3 Exoenzyme

Studies were performed to determine the unknown status of PKC and RhoA/ROCK in the phorbol 12,13-dibutyrate (PDBu)-stimulated state in the human internal anal sphincter (IAS) smooth muscle cells (SMCs). We determined the effects of PDBu (10−7M), the PKC activator, on PKCα and RhoA and ROCK II translocation in the human IAS SMCs. We used immunocytochemistry and fluorescence microcopy in the basal state, following PDBu, and before and after PKC inhibitor calphostin C (10−6M), cell-permeable RhoA inhibitor C3 exoenzyme (2.5 μg/ml), and ROCK inhibitor Y 27632 (10−6M). We also determined changes in the SMC lengths via computerized digital micrometry. In the basal state PKCα was distributed almost uniformly throughout the cell, whereas RhoA and ROCK II were located in the higher intensities toward the periphery. PDBu caused significant translocation of PKCα, RhoA, and ROCK II. PDBu-induced translocation of PKCα was attenuated by calphostin C and not by C3 exoenzyme and Y 27632. However, PDBu-induced translocation of RhoA was blocked by C3 exoenzyme, and that of ROCK II was attenuated by both C3 exoenzyme and Y 27632. Contraction of the human IAS SMCs caused by PDBu in parallel with RhoA/ROCK II translocation was attenuated by C3 exoenzyme and Y 27632 but not by calphostin C. In human IAS SMCs RhoA/ROCK compared with PKC are constitutively active, and contractility by PDBu is associated with RhoA/ROCK activation rather than PKC. The relative contribution of RhoA/ROCK vs. PKC in the pathophysiology and potential therapy for the IAS dysfunction remains to be determined.

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