Brian A. Perrino

Novel regulation of the A-type K+ current in murine proximal colon by calcium-calmodulin-dependent protein kinase II

1 The kinetics of inactivation of delayed rectifier K+ current in murine colonic myocytes differed in amphotericin‐permeabilized patch and conventional patch clamp. The difference was accounted for by Ca2+ buffering. 2 Calcium‐calmodulin‐dependent protein kinase II (CaMKII) inhibitors increased the rate of inactivation and slowed recovery from inactivation of the outward current. This was seen in single steps and in the envelope of the current tails. The effect was largely on the TEA‐insensitive component of current. 3 Dialysis of myocytes with autothiophosphorylated CaMKII slowed inactivation. This effect was reversed by addition of CaMKII inhibitor. 4 Antibodies revealed CaMKII‐like immunoreactivity in murine colonic myocytes and other cells. Immunoblots identified a small protein with CaMKII‐like immunoreactivity in homogenates of colonic muscle. 5 We conclude that CaMKII regulates delayed rectifier K+ currents in murine colonic myocytes. The changes in the delayed rectifier current may participate in the Ca2+‐dependent regulation of gastrointestinal motility.

Ca2+ sensitization pathways accessed by cholinergic neurotransmission in the murine gastric fundus

•  In smooth muscles, the sensitivity of contraction to Ca2+ can be increased by the phosphorylation of CPI‐17 and MYPT1, resulting in the inhibition of myosin light chain phosphatase (MLCP). •  Ca2+ sensitization of smooth muscle contraction has typically been studied by immersing muscles in solutions containing contractile agonists. •  However, stimulating muscles by bath‐applied agonists may not be equivalent to neurotransmitter release because different post‐junctional receptors may be activated in response to these different modes of stimulation. •  In this study we found that a bath‐applied cholinergic agonist activates Ca2+ sensitization mechanisms in gastric fundus smooth muscles that are different than those of cholinergic neurotransmission. Electrical field stimulation (EFS) only increased CPI‐17 phosphorylation, while bath‐applied carbachol increased both CPI‐17 and MYPT1 phosphorylation. •  With the cholinesterase inhibitor neostigmine present, both CPI‐17 and MYPT1 phosphorylation were increased by EFS. •  In fundus muscles of W/Wv mice which lack intramuscular interstitial cells of Cajal (ICC‐IMs), EFS alone increased both CPI‐17 and MYPT1 phosphorylation. •  These findings indicate that ACh availability determines which Ca2+ sensitization mechanisms are activated, and ICC‐IMs regulate the access of ACh to smooth muscles.

Regulation of SRF/CArG-dependent gene transcription during chronic partial obstruction of murine small intestine.

Abstract  Intestinal obstructions lead to a variety of motility disorders. Small intestine smooth muscles undergo dramatic phenotypic changes in response to obstruction, but the underlying molecular mechanisms are unknown. Using RT‐PCR, ChIP, Re‐ChIP, and Western blots, we examined the effect of small bowel mechanical obstruction on smooth muscle gene expression. Obstruction caused a transient hyperplasia, followed by a prolonged hypertrophic response of small intestine smooth muscle cells. Smooth muscle myosin heavy chain (MHC), α‐actin, and γ‐actin expression decreased initially, and then increased as hypertrophy developed. Myocardin expression decreased initially and then increased, while kruppel‐like factors (KLF)4 and KLF5 expression increased initially, and then decreased. Serum response factor (SRF) expression decreased initially, and then recovered to sham‐operated levels as hypertrophy developed. SRF binding to smooth muscle MHC and α‐actin promoters decreased initially, but then increased above sham‐operated levels as hypertrophy developed. Elk‐1 binding to smooth muscle myosin heavy chain and α‐actin promoters increased initially, and then decreased to sham‐operated levels as hypertrophy developed. c‐fos expression increased initially, which was associated with increased SRF/Elk‐1 binding to the c‐fos promoter. The Elk‐1 phosphorylation inhibitor U‐0126 inhibited the increase in c‐fos expression. These findings indicate a dynamic response of small intestine smooth muscles to bowel obstruction involving switching between differentiated, proliferative, and hypertrophic phenotypes. These results suggest that changes in the expression and interactions between SRF, myocardin, Elk‐1, and c‐fos play key roles in the phenotypic switching of small intestine smooth muscles in response to mechanical obstruction.

Activation of calcineurin A subunit phosphatase activity by its calcium-binding B subunit.

The protein phosphatase activity of calcineurin (CaN) is activated through calcium binding to both calmodulin and the B subunit of CaN. The purpose of this study was to determine which domain(s) in the CaN B subunit is required for either binding to the CaN A subunit or for transducing the effects of B subunit Ca2+ binding to the stimulation of the CaN A subunit phosphatase activity. We have previously demonstrated that interaction of CaN B regulatory subunit with the CaN A catalytic subunit requires hydrophobic residues within the CaN A sequence 328-390 [Watanabe Y., Perrino, B.A., Chang, B.H., & Soderling, T.R. (1995) J. Biol. Chem. 270, 456-460]. In the present study, selected hydrophobic residues within the B subunit were mutated to Glu to Gln. CaN B subunit mutants BE-1 (Val115/Leu116 to Glu), BE-2 (Val156/157/168/169 to Glu), and BQ-2 (Val156/157/168/169 to Gln) were expressed and purified. The three mutant B subunits bound 45Ca2+ normally. Mutants BE-2 and BQ-2 interacted with a GST fusion protein containing the B subunit binding domain of the CaN A subunit (residues 328-390), and they stimulated the phosphatase activity of the CaN A subunit in an in vitro reconstitution assay. Mutant BE-1 had a 3-fold reduced affinity for binding CaN A, and this mutant, even at saturating concentrations, gave very little stimulation of CaN A phosphatase activity. We conclude that residues Val115/Leu116 in the B subunit participate in high-affinity binding to the A subunit and are required for transducing the effects [i.e., decrease Km and increase Vmax; Perrino, B.A., Ng, L.Y., & Soderling, T.R. (1995) J. Biol. Chem. 270, 340-346] of B subunit Ca2+ binding to stimulation of CaN A phosphatase activity.

Regulation of basal LC20 phosphorylation by MYPT1 and CPI-17 in murine gastric antrum, gastric fundus, and proximal colon smooth muscles

Background  Myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) govern myosin light chain (LC20) phosphorylation and smooth muscle contraction. Rho kinase (ROK) inhibits MLCP, resulting in greater LC20 phosphorylation and force generation at a given [Ca2+]i. Here, we investigate the role of ROK in regulating LC20 phosphorylation and spontaneous contractions of gastric fundus, gastric antrum, and proximal colon smooth muscles.

Inhibition of BKCa channel activity by association with calcineurin in rat brain

Large conductance calcium‐activated potassium (BKCa) channels are regulated by a number of different protein kinases and phosphatases. The close association of enzymes and channel have been shown to underlie many examples of modulation. However, only the association of protein kinase A with the BKCa channel has been detailed [ Tian et al. (2003)J. Biol. Chem., 278, 8669–8677]. We have found using reciprocal immunoprecipitations that the BKCa channel associates with the calcium/calmodulin‐dependent phosphatase calcineurin, in Wistar rat brain. A HA‐tagged construct of the carboxyl terminus of rSlo27, a variant of the BKCa channel that is abundant in the hippocampus [ Ha et al. (2000)Eur. J. Biochem., 267, 910–9218], was found to associate only with the B subunit of calcineurin. This data suggests that the majority of the interaction of the BKCa channel with calcineurin is mediated by the B subunit of the phosphatase. This was confirmed by using glutathione‐S‐transferase (GST) fusion proteins of the linker regions between the S7–S10 hydrophobic domains in the carboxyl terminus of rSlo27, where only the B subunit of calcineurin interacted with regions between S7 and S9 of the channel. Addition of a constitutively active calcineurin (CaN420) to inside‐out membrane patches excised from cultured hippocampal neurons resulted in a dramatic reduction in BKCa channel open probability, with only very short‐duration events being apparent. These data suggest that BKCa channel activity is inhibited by calcineurin, an effect mediated by the association of the calcineurin B subunit with the carboxyl terminus of the channel.

Tra2β Protein Is Required for Tissue-specific Splicing of a Smooth Muscle Myosin Phosphatase Targeting Subunit Alternative Exon

Background: Alternative splicing of MYPT1 E23 defines fast versus slow smooth muscle. Results: Tra2β is required for the splicing of Mypt1 E23 in fast smooth muscle. Conclusion: Tra2β splicing factor confers unique contractile properties to fast smooth muscle. Significance: This is the first identification of a gene regulatory pathway conferring sensitivity to cGMP signaling in smooth muscle. Alternative splicing of the smooth muscle myosin phosphatase targeting subunit (Mypt1) exon 23 (E23) is tissue-specific and developmentally regulated and, thus, an attractive model for the study of smooth muscle phenotypic specification. We have proposed that Tra2β functions as a tissue-specific activator of Mypt1 E23 splicing on the basis of concordant expression patterns and Tra2β activation of Mypt1 E23 mini-gene splicing in vitro. In this study we examined the relationship between Tra2β and Mypt1 E23 splicing in vivo in the mouse. Tra2β was 2- to 5-fold more abundant in phasic smooth muscle tissues, such as the portal vein, small intestine, and small mesenteric artery, in which Mypt1 E23 is predominately included as compared with the tonic smooth muscle tissues, such as the aorta and inferior vena cava, in which Mypt1 E23 is predominately skipped. Tra2β was up-regulated in the small intestine postnatally, concordant with a switch to Mypt1 E23 splicing. Targeting of Tra2β in smooth muscle cells using SM22α-Cre caused a substantial reduction in Mypt1 E23 inclusion specifically in the intestinal smooth muscle of heterozygotes, indicating sensitivity to Tra2β gene dosage. The switch to the Mypt1 E23 skipped isoform coding for the C-terminal leucine zipper motif caused increased sensitivity of the muscle to the relaxant effects of 8-Br-cyclic guanosine monophosphate (cGMP). We conclude that Tra2β is necessary for the tissue-specific splicing of Mypt1 E23 in the phasic intestinal smooth muscle. Tra2β, by regulating the splicing of Mypt1 E23, sets the sensitivity of smooth muscle to cGMP-mediated relaxation.

Responses to Enteric Motor Neurons in the Gastric Fundus of Mice With Reduced Intramuscular Interstitial Cells of Cajal

Background/Aims Interstitial cells of Cajal (ICC) play important functions in motor activity of the gastrointestinal tract. The role of ICC as pacemakers is well established, however their participation in neurotransmission is controversial. Studies using mutant animals that lack ICC have yielded variable conclusions on their importance in enteric motor responses. The purpose of this study was to: (1) clarify the role of intramuscular ICC (ICC-IM) in gastric motor-neurotransmission and (2) evaluate remodeling of enteric motor responses in W/WV mice. Methods Kit immunohistochemistry and post-junctional contractile responses were performed on fundus muscles from wild-type and W/WV mice and quantitative polymerase chain reaction (qPCR) was used to evaluate differences in muscarinic and neurokinin receptor expression. Results Although ICC-IM were greatly reduced in comparison with wild-type mice, we found that ICC-IM persisted in the fundus of many W/WV animals. ICC-IM were not observed in W/WV group 1 (46%) but were observed in W/WV group 2 (40%). Evoked neural responses consisted of excitatory and inhibitory components. The inhibitory component (nitrergic) was absent in W/WV group 1 and reduced in W/WV group 2. Enhanced excitatory responses (cholinergic) were observed in both W/WV groups and qPCR revealed that muscarinic-M3 receptor expression was significantly augmented in the W/WV fundus compared to wild-type controls. Conclusions This study demonstrates that ICC-IM mediate nitrergic inhibitory neurotransmission in the fundus and provides evidence of plasticity changes in neuronal responses that may explain discrepancies in previous functional studies which utilized mutant animals to examine the role of ICC-IM in gastric enteric motor responses.

CaM kinase II in colonic smooth muscle contributes to dysmotility in murine DSS-colitis

Background  Altered calcium mobilization has been implicated in the development of colonic dysmotility in inflammatory bowel disease. The aim of this study was to investigate the mechanisms by which disrupted intracellular Ca2+ signalling contributes to the impaired contractility of colon circular smooth muscles.

Calcium Sensitization Mechanisms in Gastrointestinal Smooth Muscles.

An increase in intracellular Ca2+ is the primary trigger of contraction of gastrointestinal (GI) smooth muscles. However, increasing the Ca2+ sensitivity of the myofilaments by elevating myosin light chain phosphorylation also plays an essential role. Inhibiting myosin light chain phosphatase activity with protein kinase C-potentiated phosphatase inhibitor protein-17 kDa (CPI-17) and myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation is considered to be the primary mechanism underlying myofilament Ca2+ sensitization. The relative importance of Ca2+ sensitization mechanisms to the diverse patterns of GI motility is likely related to the varied functional roles of GI smooth muscles. Increases in CPI-17 and MYPT1 phosphorylation in response to agonist stimulation regulate myosin light chain phosphatase activity in phasic, tonic, and sphincteric GI smooth muscles. Recent evidence suggests that MYPT1 phosphorylation may also contribute to force generation by reorganization of the actin cytoskeleton. The mechanisms responsible for maintaining constitutive CPI-17 and MYPT1 phosphorylation in GI smooth muscles are still largely unknown. The characteristics of the cell-types comprising the neuroeffector junction lead to fundamental differences between the effects of exogenous agonists and endogenous neurotransmitters on Ca2+ sensitization mechanisms. The contribution of various cell-types within the tunica muscularis to the motor responses of GI organs to neurotransmission must be considered when determining the mechanisms by which Ca2+ sensitization pathways are activated. The signaling pathways regulating Ca2+ sensitization may provide novel therapeutic strategies for controlling GI motility. This article will provide an overview of the current understanding of the biochemical basis for the regulation of Ca2+ sensitization, while also discussing the functional importance to different smooth muscles of the GI tract.