Caroline A Cobine

University of Nevada School of Medicine, Reno, Nevada, United States

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Publications (9)47.23 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The effector cells and second messengers participating in nitrergic neuromuscular transmission (NMT) were investigated in the mouse internal anal sphincter (IAS). Protein expression of guanylate cyclase (GCα, GCβ) and cyclic GMP dependent protein kinase I (cGKI) were examined in cryostat sections with dual labeling immunohistochemical techniques in PDGFRα(+) cells, interstitial cells of Cajal (ICC) and smooth muscle cells (SMC). Gene expression levels were determined with qPCR of dispersed cells from Pdgfrα(egfp/+), Kit(copGFP/+) and smMHC(Cre-egfp) mice sorted with FACS. The relative gene and protein expression levels of GCα and GCβ were: PDGFRα(+) cells>ICC>SMC. In contrast, cGKI gene expression sequence was SMC=ICC>PDGFRα(+) cells while cGKI protein expression sequence was neurons>SMC>ICC=PDGFRα(+) cells. The functional role of cGKI was investigated in cGKI(-/-) mice. Relaxation with 8-Br-cGMP was greatly reduced in cGKI(-/-) mice while responses to sodium nitroprusside (SNP) were partially reduced and forskolin responses were unchanged. A nitrergic relaxation occurred with nerve stimulation (NS, 5Hz, 60s) in cGKI(+/+) and cGKI(-/-) mice although there was a small reduction in the cGKI(-/-) mouse. L-NNA abolished responses during the first 20-30s of NS in both animals. The GC inhibitor ODQ greatly reduced or abolished SNP and nitrergic NS responses in both animals. These data confirm an essential role for GC in NO-induced relaxation in the IAS. However, the expression of GC and cGKI by all three cell types suggests that each may participate in coordinating muscular responses to NO. The persistence of nitrergic NMT in the cGKI(-/-) mouse suggests the presence of a significant GC-dependent, cGKI-independent pathway.
    American journal of physiology. Gastrointestinal and liver physiology. 10/2014;
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    ABSTRACT: There is evidence that vasoactive intestinal peptide (VIP) participates in inhibitory neuromuscular transmission (NMT) in the internal anal sphincter (IAS). However, specific details concerning VIP-ergic NMT are limited largely because of difficulties in selectively blocking other inhibitory neural pathways. The present study used the selective P2Y1 receptor antagonist MRS2500 (1 μM) and the NOS inhibitor L-NNA (100 μM) to block purinergic and nitrergic NMT to characterize non-purinergic, non-nitrergic (NNNP) inhibitory NMT and the role of VIP in this response. Nerves were stimulated with electrical field stimulation (EFS; 0.1-20 Hz, 4-60s) and the associated changes in contractile and electrical activity measured under non-adrenergic, non-cholinergic conditions in the IAS of wildtype (WT) and VIP-/- mice. EFS gave rise to frequency dependent relaxation and hyperpolarization that was blocked by tetrodotoxin. Responses during brief trains of stimuli (4s) were mediated by purinergic and nitrergic NMT. During longer stimulus trains a NNNP relaxation and hyperpolarization developed slowly and persisted for several minutes beyond the end of the stimulus train. NNNP NMT was abolished by VIP 6-28 (30 μM), absent in the VIP-/- mouse and mimicked by exogenous VIP (1-100 nM). VIP immunoreactivity was co-localized with nNOS in varicose intramuscular fibres but was not detected in the VIP-/- mouse IAS. In conclusion, this study identified an ultraslow component of inhibitory NMT in the IAS mediated by VIP. In vivo, this pathway may be activated with larger rectal distensions leading to a more prolonged period of anal relaxation.
    The Journal of Physiology 01/2013; · 4.38 Impact Factor
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    A M Duffy, C A Cobine, K D Keef
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    ABSTRACT: Intramuscular interstitial cells of Cajal (ICC-IM) have been shown to participate in nitrergic neuromuscular transmission (NMT) in various regions of the gastrointestinal (GI) tract, but their role in the internal anal sphincter (IAS) is still uncertain. Contractile studies of the IAS in the W/W(v) mouse (a model in which ICC-IM numbers are markedly reduced) have reported that nitrergic NMT persists and that ICC-IM are not required. However, neither the changes in electrical events underlying NMT nor the contributions of other non-nitrergic neural pathways have been examined in this model. The role of ICC-IM in NMT was examined by recording the contractile and electrical events associated with electrical field stimulation (EFS) of motor neurons in the IAS of wildtype and W/W(v) mice. Nitrergic, purinergic, and cholinergic components were identified using inhibitors of these pathways. Under NANC conditions, purinergic and nitrergic pathways both contribute to EFS-induced inhibitory junction potentials (IJPs) and relaxation. Purinergic IJPs and relaxation were intact in the W/W(v) mouse IAS, whereas nitrergic IJPs were reduced by 50-60% while relaxation persisted. In the presence of L-NNA (NOS inhibitor) and MRS2500 (P2Y1 receptor antagonist), EFS gave rise to cholinergic depolarization and contractions that were abolished by atropine. Cholinergic depolarization was absent in the W/W(v) mouse IAS while contraction persisted. ICC-IM significantly contributes to the electrical events underlying nitrergic and cholinergic NMT, whereas contractile events persist in the absence of ICC-IM. The purinergic inhibitory neural pathway appears to be independent of ICC-IM.
    Neurogastroenterology and Motility 11/2011; 24(1):e41-55. · 2.94 Impact Factor
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    ABSTRACT: Interstitial cells of Cajal (ICC) have been shown to participate in nitrergic neurotransmission in various regions of the gastrointestinal (GI) tract. Recently, fibroblast-like cells, which are positive for platelet-derived growth factor receptor α (PDGFRα(+)), have been suggested to participate additionally in inhibitory neurotransmission in the GI tract. The distribution of ICC and PDGFRα(+) cell populations and their relationship to inhibitory nerves within the mouse internal anal sphincter (IAS) are unknown. Immunohistochemical techniques and confocal microscopy were therefore used to examine the density and arrangement of ICC, PDGFRα(+) cells and neuronal nitric-oxide-synthase-positive (nNOS(+)) nerve fibers in the IAS of wild-type (WT) and W/W ( v ) mice. Of the total tissue volume within the IAS circular muscle layer, 18% consisted in highly branched PDGFRα(+) cells (PDGFRα(+)-IM). Other populations of PDGFRα(+) cells were observed within the submucosa and along the serosal and myenteric surfaces. Spindle-shaped intramuscular ICC (ICC-IM) were present in the WT mouse IAS but were largely absent from the W/W ( v ) IAS. The ICC-IM volume (5% of tissue volume) in the WT mouse IAS was significantly smaller than that of PDGFRα(+)-IM. Stellate-shaped submucosal ICC (ICC-SM) were observed in the WT and W/W ( v ) IAS. Minimum surface distance analysis revealed that nNOS(+) nerve fibers were closely aligned with both ICC-IM and PDGFRα(+)-IM. An even closer association was seen between ICC-IM and PDGFRα(+)-IM. Thus, a close morphological arrangement exists between inhibitory motor neurons, ICC-IM and PDGFRα(+)-IM suggesting that some functional interaction occurs between them contributing to inhibitory neurotransmission in the IAS.
    Cell and Tissue Research 02/2011; 344(1):17-30. · 3.68 Impact Factor
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    ABSTRACT: The morphology of interstitial cells of Cajal (ICC) in the circular muscle layer of the cynomolgus monkey internal anal sphincter (IAS) and rectum and their relationship to sympathetic and nitrergic nerves were compared by dual-labeling immunohistochemistry. Contractile studies confirmed that nitrergic nerves participate in neural inhibition in both regions whereas sympathetic nerves serve as excitatory motor nerves only in the IAS. Muscle bundles extended from myenteric to submucosal edge in rectum but in the IAS bundles were further divided into "minibundles" each surrounded by connective tissue. Dual labeling of KIT and smooth muscle myosin revealed KIT-positive stellate-shaped ICC (ICC-IAS) within each minibundle. In the rectum intramuscular ICC (ICC-IM) were spindle shaped whereas stellate-shaped ICC were located at the myenteric surface (ICC-MY). ICC were absent from both the myenteric and submucosal surfaces of the IAS. Nitrergic nerves (identified with anti-neuronal nitric oxide synthase antibodies or NADPH diaphorase activity) and sympathetic nerves (identified with anti-tyrosine hydroxylase antibody) each formed a plexus at the myenteric surface of the rectum but not the IAS. Intramuscular neuronal nitric oxide synthase- and tyrosine hydroxylase-positive fibers were present in both regions but were only closely associated with ICC-IM in rectum. Minimal association was also noted between ICC-IAS and cells expressing the nonspecific neuronal marker PGP9.5. In conclusion, the morphology of rectal ICC-IM and ICC-MY is similar to that described elsewhere in the gastrointestinal tract whereas ICC-IAS are unique. The distribution of stellate-shaped ICC-IAS throughout the musculature and their absence from both the myenteric and submucosal surfaces suggest that ICC-IAS may serve as pacemaker cells in this muscle whereas their limited relationship to nerves suggests that they are not involved in neuromuscular transmission. Additionally, the presence of numerous minibundles, each containing both ICC-IAS and nerves, suggests that this muscle functions as a multiunit type muscle.
    AJP Gastrointestinal and Liver Physiology 02/2010; 298(5):G643-56. · 3.65 Impact Factor
  • Gastroenterology 01/2008; 134(4). · 12.82 Impact Factor
  • Gastroenterology 01/2008; 134(4). · 12.82 Impact Factor
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    C A Cobine, M Fong, R Hamilton, K D Keef
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    ABSTRACT: Excitatory motor innervation to the internal anal sphincter (IAS) of the monkey, the rabbit and mouse were compared. Contractile responses to electrical field stimulation of nerves (EFS, atropine 1 micromol L(-1) and N(omega)-nitro-L-arginine 100 micromol L(-1) present throughout) were examined in isolated strips of IAS. In the monkey IAS, EFS caused frequency dependent (1-30 Hz) contractions which were abolished by guanethidine (10 micromol L(-1)) or phentolamine (3 micromol L(-1)). The sympathetic neurotransmitter noradrenaline (NA) also caused concentration-dependent (10 nmol L(-1)-100 micromol L(-1)) contractions which were abolished by phentolamine revealing a small relaxation that was abolished by propranolol (3 micromol L(-1)). In contrast, EFS caused only relaxation of the mouse and rabbit IAS which was not affected by guanethidine. Furthermore, NA relaxed these muscles and relaxation was nearly abolished by combined addition of phentolamine and propranolol. In conclusion, the monkey IAS is functionally innervated by sympathetic nerves that contract the muscle via excitatory alpha-adrenergic receptors. In contrast, no significant motor function could be identified for sympathetic nerves in the rabbit or mouse IAS although adrenergic receptors linked to muscle inhibition are present. These data reveal species dependent differences in sympathetic motor innervation and suggest that some species are more appropriate than others as models for motor innervation to the human IAS.
    Neurogastroenterology and Motility 12/2007; 19(11):937-45. · 2.94 Impact Factor
  • Caroline A Cobine, Brid P Callaghan, Kathleen D Keef
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    ABSTRACT: The present study investigated active tone development in isolated ring segments of rabbit epicardial coronary artery. Endothelium-denuded (E-) or endothelium-intact (E+) vessels treated with the NO synthase inhibitor N(omega)-nitro-L-arginine (100 microM) developed active tone, which was enhanced by stretch and reversed by the NO donor sodium nitroprusside (SNP; IC(50)=9 nM). Nifedipine abolished active tone and the contractile response to phorbol dibutyrate (PDBu; 10 nM) with the same potency (IC(50)=8 nM), whereas 300 nM PDBu responses were only partially blocked by nifedipine. The classical and novel PKC inhibitors GF-109203X (IC(50)=1-2 microM) and chelerythrine (IC(50)=4-5 microM) and the classical PKC inhibitor Gö-6976 (IC(50)=0.3-0.4 microM) blocked both active tone and 10 nM PDBu responses with similar potency. Active tone development was associated with depolarization of membrane potential (E(m)) and a shift to the left of the E(m)-vs.-contraction relationship determined by varying extracellular potassium. The depolarization and leftward shift were reversed by either chelerythrine (10 microM) or SNP (30 nM). PDBu (100-300 nM) increased peak L-type calcium channel (Ca(v)) currents in isolated coronary myocytes, and this effect was reversed by chelerythrine (1 microM) or Gö-6976 (200 nM). SNP (500 nM) reduced Ca(v) currents only in the presence of the PKA blocker 8-bromo-2'-O-monobutyryl-cAMPS, Rp isomer (10 microM). In conclusion, active tone development in coronary artery is suppressed by basal NO release and is dependent on both enhanced Ca(v) activity and classical PKC activity. Both E(m)-dependent and -independent processes contribute to contraction. Our results suggest that one E(m)-independent process is direct enhancement of Ca(v) current by PKC.
    AJP Heart and Circulatory Physiology 07/2007; 292(6):H3079-88. · 4.01 Impact Factor