Biomedical subjects

Hamid I Akbarali

Publications and source records attributed to Hamid I Akbarali.

8 recordsLinked to original sources

PubMed2004

Modulation of TRPV1 by nonreceptor tyrosine kinase, c-Src kinase.

The capsaicin receptor TRPV1 is a nonselective cation channel that is expressed in sensory neurons. In this study, we examined the role of the nonreceptor cellular tyrosine kinase c-Src kinase in the modulation of the rat TRPV1. Capsaicin-induced currents in identified colonic dorsal root ganglion neurons were blocked by the c-Src kinase inhibitor PP2 and enhanced by the tyrosine phosphatase inhibitor sodium orthovandate. PP2 also abolished currents in human embryonic kidney-293 cells transfected with rat TRPV1, whereas cotransfection of TRPV1 with v-Src resulted in fivefold increase in capsaicin-induced currents. In cells transfected with dominant-negative c-Src and TRPV1, capsaicin-induced currents were decreased by approximately fourfold. TRPV1 co-immunoprecipitated with Src kinase and was tyrosine phosphorylated. These studies demonstrate that TRPV1 is a potential target for cellular tyrosine kinase-dependent phosphorylation.

Animals↗

PubMed2004

Altered gene expression and increased bursting activity of colonic smooth muscle ATP-sensitive K+ channels in experimental colitis.

The ATP-sensitive K(+) channel (K(ATP)) is a complex composed of an inwardly rectifying, pore-forming subunit (Kir 6.1 and Kir 6.2) and the sulfonylurea receptor (SUR1 and SUR2). In gastrointestinal smooth muscle, these channels are important in regulating cell excitability. We examined the molecular composition of the K(ATP) channel in mouse colonic smooth muscle and determined its activity in the pathophysiological setting of experimental colitis. Following 7 days of dextran sulfate sodium (DSS) treatment in drinking water, colonic inflammation was scored by histology and physical signs. In whole cell recordings, levcromakalim-induced currents were significantly larger in inflamed cells. In cell-attached patch recordings of single-channel events, levcromakalim enhanced the bursting duration in inflamed cells. The single-channel conductance of approximately 42 pS was not altered with inflammation. mRNA for both Kir 6.1 and 6.2 were detected by RT-PCR. Kir 6.1 was localized to the plasma membrane, whereas Kir 6.2 was mainly detected in the cytosol by immunohistochemistry. Quantitative PCR showed that Kir 6.1 gene expression was upregulated by almost 22-fold, whereas SUR2B was downregulated by threefold after inflammation. Thus decreased motility of the colon during inflammation may be associated with changes in the transcriptional regulation of Kir 6.1 and SUR2B gene expression.

ATP-Binding Cassette Transporters↗

PubMed2004

Protein and gene expression of Ca2+ channel isoforms in murine colon: effect of inflammation.

L-Type voltage-dependent Ca2+ channels (L-VDCC) mediate calcium influx in response to membrane depolarization and regulate intracellular processes such as contraction, secretion, neurotransmission, and gene expression. Colonic inflammation significantly attenuates calcium currents in smooth muscle; however, the basis for this remains unclear. In this study we examined the protein and mRNA expression of two isoforms of Ca(v)1.2, encoded by either exon la or 1b. Both isoforms were detected by Western blots, immunohistochemistry and RT-PCR in smooth muscle cells. Neither the protein nor mRNA expression measured by real-time PCR of either isoforms was affected in colonic myocytes from dextran sulfate sodium-treated mice. In whole-cell voltage-clamp experiments, the amplitude of the calcium currents were decreased by almost 70% by inflammation. The calcium channel currents were attenuated by 50 +/- 3% by the c-src kinase specific inhibitor, PP2, in control cells but only 19 +/- 7% in cells from inflamed mice. These studies suggest that decreased calcium channel currents following colonic inflammation are not due to decreased expression but may result from altered regulation by the non-receptor cellular tyrosine kinase, c-src kinase.

Animals↗

PubMed2004

Inflammation-induced "channelopathies" in the gastrointestinal smooth muscle.

Inflammation markedly alters the motility patterns of the gastrointestinal tract, resulting mostly in decreased excitability of smooth muscle. There is emerging evidence indicating that inflammation alters ion channel expression and function of smooth muscle cells. In this review we summarize studies defining the mechanisms affecting contractile and electrical activity of gastrointestinal smooth muscle. We have focused on the evidence for decreased calcium channel conductance and alterations in the intracellular signaling mechanisms and discuss the role of muscarinic receptor activation in models of gastrointestinal inflammation. We propose that some of the clinical symptoms of altered smooth muscle contraction in pathogenesis of gut disorders such as inflammatory bowel disease may be regulated at the level of the ion channel.

Animals↗

PubMed2003

ERG K+ currents regulate pacemaker activity in ICC.

Ether-à-go-go-related gene (ERG) K channels have been implicated in the generation of pacemaker activities in the heart. To study the presence and function of ERG K channels in the pacemaker cells of the small intestine [the interstitial cells of Cajal (ICC)], a combination of patch-clamp techniques, tissue and live cell immunohistochemistry, RT-PCR, and in vitro functional studies were performed. Nonenzymatically isolated ICC in culture were identified by vital staining and presence of rhythmic inward currents. RT-PCR showed the presence of ERG mRNA in the intestinal musculature, and immunohistochemistry on tissue and cultured cells demonstrated that protein similar to human ERG was concentrated on ICC in the Auerbach's plexus region. Whole cell ERG K+ currents were evoked on hyperpolarization from 0 mV (but not from -70 mV) up to -120 mV and showed strong inward rectification. The currents were inhibited by E-4031, cisapride, La3+, and Gd3+ but not by 50 microM Ba2+. The ERG K+ inward current had a typical transient component with fast activation and inactivation kinetics followed by significant steady-state current. E-4031 also inhibited tetraethylammonium (TEA)-insensitive outward current indicating that the ERG K+ current is operating at depolarizing potentials. In contrast to TEA, blockers of the ERG K+ currents caused marked increase in tissue excitability as reflected by an increase in slow-wave duration and an increase in superimposed action potential activity. In summary, ERG K channels in ICC contribute to the membrane potential and play a role in regulation of pacemaker activity of the small intestine.

Animals↗

PubMed2002

Cloning and functional characterization of the smooth muscle ether-a-go-go-related gene K+ channel. Potential role of a conserved amino acid substitution in the S4 region.

The human ether-a-go-go-related gene (HERG) product forms the pore-forming subunit of the delayed rectifier K(+) channel in the heart. Unlike the cardiac isoform, the erg K(+) channels in native smooth muscle demonstrate gating properties consistent with a role in maintaining resting potential. We have cloned the smooth muscle isoform of HERG, denoted as erg1-sm, from human and rabbit colon. erg1-sm is truncated by 101 amino acids in the C terminus due to a single nucleotide deletion in the 14th exon. Sequence alignment against HERG showed a substitution of alanine for valine in the S4 domain. When expressed in Xenopus oocytes, erg1-sm currents had much faster activation and deactivation kinetics compared with HERG. Step depolarization positive to -20 mV consistently produced a transient outward component. The threshold for activation of erg1-sm was -60 mV and steady-state conductance was approximately 10-fold greater than HERG near the resting potential of smooth muscle. Site-directed mutagenesis of alanine to valine in the S4 region of erg1-sm converted many of the properties to that of the cardiac HERG, including shifts in the voltage dependence of activation and slowing of deactivation. These studies define the functional role of a novel isoform of the ether-a-go-go-related gene K(+) channel in smooth muscle.

Amino Acid Sequence↗

PubMed2002

Fenamate-induced enhancement of heterologously expressed HERG currents in Xenopus oocytes.

The human ether-a-go-go related gene (HERG) product encodes for the pore-forming subunit of the rapid component of the delayed rectifier K(+) channel that mediates repolarization of cardiac action potential. HERG channels are also potential targets of a large variety of pharmacological agents most of which tend to block HERG currents. In this study, we examined the effects of the non-steroidal anti-inflammatory agents, flufenamic acid and niflumic acid, on heterologously expressed HERG channels in oocytes. The cRNA of HERG (30 ng) was injected into Xenopus oocytes and currents were recorded using two-electrode voltage clamp technique in a low Cl(-) solution. Flufenamic and niflumic acids (10(-4)-5 x 10 (-4) M) enhanced the amplitude of outward currents evoked by depolarizing pulses. At potentials positive to 0 mV, an initial transient component was also evident in the presence of fenamates. Fenamates accelerated the activation rate of HERG channels and decelerated their deactivation. Flufenamic acid (5 x 10 (-4) M) shifted the I(tail)-V relationship from -26.7+/-0.1 to -31.4+/-0.2 mV. Neither flufenamic acid or niflumic acid affected the kinetics of HERG channel inactivation. Using a voltage protocol that mimicked the cardiac action potential, both fenamates increased the outward current during the plateau and during the phase 3 repolarization of action potential. The effects of the fenamates were blocked by the HERG channel blocker, E-4031 and were also not observed in water-injected oocytes. Our data suggest that fenamates enhance HERG currents and affect the action potential duration in the heart.

Action Potentials↗

PubMed2002

Coupling of M2 muscarinic receptor to L-type Ca channel via c-src kinase in rabbit colonic circular smooth muscle.

BACKGROUND & AIMS: The L-type Ca(2+) channel is a major pathway for Ca(2+) influx in colonic smooth muscle and is modulated by endogenous levels of nonreceptor tyrosine kinase, c-src. Tyrosine kinases are also activated by G-protein-coupled receptors (GPCR). This study determined whether muscarinic receptor couples to Ca(2+) channels via c-src kinase. METHODS: Currents were measured in rabbit colonic smooth muscle cells and in transfected HEK293 cells by patch-clamp technique. Tyrosyl phosphorylated proteins were detected by Western blots and the interaction of c-src with the c-terminus of alpha subunit of Ca(2+) channel was determined by a GST pull-down assay. RESULTS: Methacholine (10 micromol/L) enhanced Ca(2+) channel currents by 30% under conditions whereby the M(3) receptor pathway was blocked by either 4-DAMP or by intracellular dialysis with anti-Galphaq antibody. Similar effects were observed by blocking intracellular Ca(2+) release with heparin. Enhancement was abolished by intracellular anti-Galphai antibody and by the c-src inhibitor, PP2 but unaffected by the inactive analog PP3. Immunoblot with anti-src antibody revealed increased src phosphorylation by muscarinic receptor stimulation. Purified c-src directly associated with the c-terminus of alpha1c subunit of the Ca(2+) channel. In M(2) receptor transfected HEK293 cells, currents were enhanced 2-fold by carbachol. CONCLUSIONS: These studies demonstrate stimulation of Ca(2+) current in colonic smooth muscle cells by M2 receptor coupled to Galphai-G protein and c-src activation. They also suggest a central role of c-src kinase in the cross-talk between tyrosine kinase receptor and GPCR.

Animals↗