Biomedical subjects

L W Liu

Publications and source records attributed to L W Liu.

At least 19 recordsLinked to original sources

PubMed2001

Muscarinic activation of transient inward current and contraction in canine colon circular smooth muscle cells.

Muscarinic receptor mediated membrane currents and contractions were studied in isolated canine colon circular smooth muscle cells. Carbachol (10(-5) M) evoked a slow transient inward current that was superimposed by a transient outward current at holding potentials greater than -50 mV. Carbachol contracted the cells by 70 +/- 2%. The effects of carbachol were blocked by atropine (10(-6) M), tetraethyl ammonium (20 mM), and BAPTA-AM (25 mM applied for 20 min). The inward current and contraction were not sensitive to diltiazem (10(-5) M), nitrendipine (3 x 10(-7) M), niflumic acid (10(-5) M), or N-phenylanthranilic acid (10(-4) M), but were gradually inhibited after repetitive stimulations in Ca2+ free solution. Ni2+ (2 mM) inhibited the inward current by 67 +/- 4%. The inward current reversed at +15 mV. The outward component could be selectively inhibited by iberiotoxin (20 nM) or by intracellular Cs+. Repeated stimulation in the presence of cyclopiazonic acid (CPA, 3 microM) inhibited the carbachol-induced outward current and partially inhibited contraction. CPA did not inhibit the inward current. In conclusion, muscarinic receptor stimulation evoked a CPA-sensitive calcium release that caused contraction and a CPA-insensitive transient inward current was activated that is primarily carried by Ca2+ ions and is sensitive to Ni2+.

Animals↗

PubMed1999

Mus and Peromyscus chromosome homology established by FISH with three mouse paint probes.

Fluorescence-labeled DNA probes constructed from three whole house mouse (Mus domesticus) chromosomes were hybridized to metaphase spreads from deer mouse (Peromyscus maniculatus) to identify homologies between the species. Mus Chr 7 probe hybridized strongly to the ad-centromeric two-thirds of Peromyscus Chr 1q. Most of Mus 3 probe hybridized principally to two disjunct segments of Peromyscus Chr 3. Mus Chr 9 probe hybridized entirely to the whole Peromyscus Chr 7. Three Peromyscus linkage groups were assigned to chromosomes, based on linkage homology with Mus. The data also are useful in interpretation of chromosomal evolutionary history in myomorphic rodents.

Animals↗

PubMed1998

Development of pacemaker activity and interstitial cells of Cajal in the neonatal mouse small intestine.

Intestinal motor patterns are not well developed in premature infants. Similarly, in neonatal mice, irregular motor patterns were observed. Pacemaker cells, identified in the small intestine as interstitial cells of Cajal (ICCs) associated with Auerbach's plexus (ICC-APs), contribute to the generation of peristaltic movements. The objective of the present study was to assess the hypothesis that abnormal gut motor activity in (preterm) newborns can be associated with underdeveloped ICCs. Specifically, the aim was to identify at which point the electrical pacemaker activity is fully developed and whether or not the development of pacemaker activity has a structural correlation with the developmental stage of ICCs. Pacemaker activity was identified as that component of the slow wave that is insensitive to L-type calcium (Ca2+) channel blockers and displays a characteristic reduction in frequency in the presence of cyclopiazonic acid (CPA), a specific inhibitor of the endoplasmic reticulum Ca2+ pump. In newborn, unfed neonates, action potentials occurred that were irregular in frequency and amplitude and sensitive to verapamil. CPA (5 microM) abolished all action potentials. Quiescent spots were observed in approximately 50% of impalements. Six hours after birth, slow-wave activity appeared at a regular frequency and amplitude, and a well-defined plateau phase was observed. Verapamil did not affect the frequency, 5 microM CPA decreased it. The effect of CPA on the pacemaker frequency 2 days after birth was identical to that observed in adult mice. In 2-hr-old neonates, ICCs could be identified through selective uptake of methylene blue, but ultrastructural features were not fully developed. At 48 hr, a complete ICC network covering Auerbach's plexus was formed, confirmed by electron microscopy. In summary, the pacemaker component of the slow waves can be identified in neonates as early as 6 hr after birth. The pacemaker component was fully developed 2 days after birth. These electrophysiological observations correlated with the development of full network characteristics of ICC-APs and the development of fully differentiated ICC-APs from "blast-like" cells.

Animals↗

PubMed1998

Interstitial cells of Cajal: mediators of communication between circular and longitudinal muscle layers of canine colon.

The network of interstitial cells of Cajal associated with Auerbach's (myenteric) plexus in the canine colon was investigated to determine its role in facilitating communication between circular and longitudinal muscle layers. Electrical coupling between the muscle layers was demonstrated by propagating extracellularly evoked electrotonic pulses from circular muscle cells to nearby longitudinal muscle cells. The likelihood of cytoplasmic continuity across Auerbach's plexus was further demonstrated by the ability of neurobiotin to spread between the interstitial cells and the circular and longitudinal muscle cells. Importantly, direct neurobiotin spread between circular and longitudinal muscle cells was not observed even when they were in close proximity as determined by confocal microscopy. When neurobiotin did spread across the two muscle layers, the intervening interstitial cells were always neurobiotin-positive. In regions where circular and longitudinal muscle cells approach each other closely, electron microscopy revealed the presence of close appositions between interstitial cells and smooth muscle cells. Gap junctions between interstitial cells and smooth muscle cells of both layers, as judged by electron microscopy, were extremely rare. Neither gap junctions nor close appositions were observed between longitudinal and circular muscle cells. The special arrangement for electrotonic coupling across Auerbach's plexus through interstitial cells of Cajal suggests controlled coupling between the two muscle layers, explaining the preservation of their distinct electrical activities.

Animals↗

PubMed1997

Circular muscle lamellae of canine colon are electrically isolated functional units.

The circular muscle (CM) layer of canine colon consists of circumferentially oriented lamellae separated by connective tissue septa. These lamellae facilitate circumferential ring contractions. Communication between CM lamellae is necessary to generate coordinated, propulsive phasic contractions to create peristaltic movement. Potential roles of the submuscular network of interstitial cells of Cajal and branching smooth muscle cells (ICC-bSM), the myenteric interstitial cells of Cajal network (ICC-AP), and the longitudinal muscle (LM) layer in mediating communication between the CM lamellae were studied by simultaneously recording with three surface electrodes, using different types of muscle strip preparations. When the ICC-bSM network was intact, slow waves were observed to be entrained both along and across CM lamellae. In contrast, the CM layer devoid of the ICC-bSM network, the myenteric plexus, and the longitudinal muscle (CM preparation) was spontaneously quiescent. Spike-like action potentials, evoked in the CM preparations by Ba2+ (0.5 mM), were entrained within CM lamellae but were not coordinated between the CM lamellae. In the LM-CM preparations, in which the longitudinal muscle and the ICC-AP network were intact, the Ba(2+)-evoked action potentials were again not coordinated across septa but entrained within CM lamellae. In a step preparation, in which the ICC-bSM network was removed from part of the muscle strip, slow waves were observed to be entrained in areas with and without the ICC-bSM network when electrodes were positioned along septa. When electrodes were positioned across CM lamellae, synchronized slow wave activity was observed only in areas with the intact ICC-bSM network and quiescent activity was recorded in areas devoid of the ICC-bSM network. These results demonstrate that CM cells are electrically coupled within a CM lamella, but not between CM lamellae. The submuscular ICC-bSM network, but not longitudinal or circular muscle cells, nor the ICC-AP, mediates communication between CM lamellae.

Animals↗

PubMed1996

Morphologic characterization of polyvinyl sponge (Ivalon) breast prosthesis.

BACKGROUND: Although almost all breast implants are made of silicon, some implants, especially the ones used in early augmentation mammoplasty, were made of other materials, one of which is polyvinyl alcohol (commercially known as the Ivalon sponge). The morphology of this type of breast implant and its associated tissue reactions have not been characterized in detail. MATERIALS AND METHODS: A pair of polyvinyl breast prostheses implanted 40 years ago in a 66-year-old woman were removed together with their capsules to correct progressive disfiguration. The implants and capsules were radiographed. Sections from these specimens were subjected to routine histologic studies and special stains, including periodic acid-Schiff and Masson's trichrome stains. RESULTS: The breast implants were composed of crystals with a pathognomonic morphology. By hematoxylin-eosin stain, these crystals were polygonal, colorless, and refractile, but nonbirefringent, and they had a characteristic bubbling internal structure. The crystals displayed a deep-blue color with Masson's trichrome stain and were strongly periodic acid-Schiff-positive, with or without diastase digestion. These crystals appeared isolated or interconnecting and were separated from one another by spaces filled with tissue fluid. The capsules were composed of the same kind of crystals, but they were heavily calcified and associated with dense fibrosis and occasional multinucleated giant cells. CONCLUSIONS: This case serves to emphasize that breast prostheses made of materials other than silicon may be rarely encountered in the surgical pathology laboratory. Although polyvinyl breast implants were abandoned, injection of polyvinyl into various tissues for therapeutic purposes is sometimes indicated. The morphologic features of polyvinyl as detailed in this study should enable prompt and accurate recognition of this material, whether it is in breast implants or other types of tissue.

Aged↗

PubMed1995

Cyclopiazonic acid, inhibiting the endoplasmic reticulum calcium pump, reduces the canine colonic pacemaker frequency.

The slow wave frequency of the canine colon has previously been hypothesized to be paced by an intracellular biochemical clock. We investigated the relationship between the endoplasmic reticulum (ER) Ca++ and the periodicity of the biochemical clock. Cyclopiazonic acid, a specific inhibitor of the ER Ca++ pump, dose-dependently decreased the pacemaker frequency. Similarly, chelating cytosolic Ca++ with bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid (BAPTA) also decreased the pacemaker frequency. These observations suggest that delaying the Ca++ uptake into the ER decreases the pacemaker frequency. The pacemaker frequency was similarly decreased by neomycin [inhibiting inositol 1,4,5-triphosphate (IP3) synthesis] and by caffeine at concentrations higher than 5 mM (inhibiting the IP3-sensitive Ca++ channels in the ER membrane). Hence the IP3-sensitive Ca++ stores are involved in the biochemical clock. Ryanodine (up to 60 microM) did not affect the pacemaker frequency, which indicates that a ryanodine-sensitive store, if it exists, is not coupled to the biochemical clock. Electron microscopy showed that the smooth ER forms an extensive network of subsurface cisternae that is closely associated with large areas of the cytoplasmic face of the plasma membrane. These structures were the most extensive in interstitial cells of Cajal, slightly less in branching smooth muscle cells and far less in circular muscle cells. In summary, on the basis of these electrophysiological and morphological observations, we hypothesize that the Ca++ refilling cycle of the IP3-sensitive calcium stores associated with the plasma membrane determines the frequency of the pacemaker activity generated by the submuscular interstitial cells of Cajal-smooth-muscle network of the canine colon.

Action Potentials↗

PubMed1994

Occupancy of anion binding exosite 2 on thrombin determines Ca2+ dependence of protein C activation.

Thrombomodulin (TM) binds thrombin to form a complex that activates the plasma anticoagulant zymogen protein C. TM is an integral membrane glycoprotein that contains a chondroitin sulfate moiety. Interaction with thrombin involves both the protein component of TM, specifically the growth factor-like repeats 4-6 (TM 4-6), and chondroitin sulfate. Removal of chondroitin sulfate decreases the affinity for thrombin approximately 10-fold and shifts the Ca2+ dependence of protein C activation from simple saturation at > or = 500 microM Ca2+ to a distinct optimum at approximately 100 microM Ca2+. Thrombin possesses two regions of high positive charge, anion binding exosites 1 and 2. Anion binding exosite 1 interacts with the growth factor region of TM while exosite 2 is involved in binding prothrombin activation fragment 2 or heparin. We demonstrate that recombinant TM, truncated at the membrane-spanning domain, or TM 4-6 can bind thrombin when fragment 2 is present either covalently attached (meizothrombin des-fragment 1) or in reversible association. With meizothrombin des-fragment 1, the Ca2+ dependence of protein C activation is independent of the presence of the chondroitin sulfate on TM. At 0.27 mM Ca2+, TM containing chondroitin sulfate binds thrombin (Kd(app) = 0.3 nM) approximately 45 times tighter than meizothrombin des-fragment 1 (Kd(app) = 14 nM). However, the chondroitin-free form binds thrombin (Kd(app) = 2.4 nM) only approximately 4 times tighter than meizothrombin des-fragment 1 (Kd(app) = 9.4 nM). These studies suggest that occupancy of anion binding exosite 2 by either chondroitin sulfate or fragment 2 alters thrombin conformation resulting in the altered Ca2+ dependence of protein C activation.

Amino Acid Sequence↗

PubMed1994

Shiftwork and heat stress in an intensive care unit.

This paper reported the results of investigation on shiftwork and heat stress in an intensive care unit. The aim of this study was to analyse the physiological strain of nurses during the three shifts (Morning, Afternoon, Night) in relation to the specific microclimatic conditions and job activities. 8 professional nurses (6 female and 2 male), aged between 21 and 38 years (mean 29.8 +/- 5.6 years), having from 3 to 18 years service, were monitored throughout a complete working cycle of 4 days. They worked on a three shift system at fast rotation. Their working environment was an independent unit for intensive care and expert surgical treatment which was entirely air conditioned. The observation indexes included: twenty-five blood parameters, ten urine parameters, net cardiac cost and relation cardiac cost, etc. The results of the investigation have not evidenced serious alterations of the psycho-physical conditions of the nurses, but do pointed out some problems pertaining both to the environmental and physiological conditions.

Adult↗

PubMed1994

Canine colonic circular muscle generates action potentials without the pacemaker component.

Two dominant types of action potentials in canine colon are slow wave type action potentials (slow waves) and spike-like action potentials (SLAPs). The slow waves, originating at the submuscular surface where a network of interstitial cells of Cajal (ICCs) is found, possess a pacemaker component. Activation of the pacemaker component is insensitive to voltage changes and L-type calcium channel blockers, and is postulated to involve a metabolic clock sensitive to cyclic AMP. SLAPs are more prominent in the longitudinal muscle. To understand the contribution circular muscle cells make to the generation of these action potentials, a circular muscle preparation (devoid of the submuscular ICC-smooth muscle network, longitudinal muscle, and myenteric plexus) was developed. Circular muscle preparations were spontaneously quiescent, with a resting membrane potential of -62.9 +/- 0.6 mV. Ba2+ (0.5 mM) depolarized the cells to -51.8 +/- 0.6 mV and induced electrical oscillations with a frequency, duration, amplitude, and rate of rise equal to 6.6 +/- 0.4 cpm, 2.2 +/- 0.2 s, 19.4 +/- 0.9 mV, and 21.8 +/- 1.7 mV/s, respectively. In most cases, Ba(2+)-induced oscillations were preceded by a prepotential of 4.4 +/- 0.3 mV, with a rate of rise of 1.1 +/- 0.1 mV/s. Ba(2+)-induced oscillations were abolished by 1 microM D600 as well as by repolarization of 6-12 mV. Addition of 0.1 microM Bay K8644 in the presence of Ba2+ further depolarized circular muscle cells to -42.4 +/- 0.8 mV and increased the oscillation frequency to 16.8 +/- 1.8 cpm. The electrical oscillations induced in circular muscle preparations by Ba2+ and Bay K8644 were similar to the SLAPs exhibited by the isolated longitudinal muscle layer, indicating that generation of SLAPs is an intrinsic property of smooth muscle cells. Forskolin (1 microM), previously shown to dramatically decrease the frequency but not the amplitude of slow waves in preparations including the submuscular ICC network, decreased the amplitude of the Ba(2+)-induced oscillations in circular muscle preparations without changing the frequency. These results provide strong evidence for the hypothesis that the submuscular ICC-smooth muscle network is essential for the initiation of the pacemaker component of the colonic slow waves. The mechanism for regulating the frequency of slow waves is different from that responsible for the Ba(2+)-induced oscillations in circular muscle preparations. Circular muscle cells are shown to be excitable and capable of generating oscillatory activity dominated by L-type calcium channel activity, which is regulated by K+ conductance.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1994

Selective lesioning of interstitial cells of Cajal by methylene blue and light leads to loss of slow waves.

Incubation with 50 microM methylene blue (MB) and subsequent intense illumination resulted in abolition of the slow-wave activity in the submuscular interstitial cells of Cajal-circular muscle (ICC-CM) preparations of canine colon. This was often accompanied by a decrease in resting membrane potential. Repolarization of cells back to -70 mV did not restore the slow-wave activity, indicating that MB plus light directly interrupted the generation mechanism of slow waves. After MB incubation, a 2-min illumination consistently changed the mitochondrial conformation in ICCs from very condensed to orthodox, without inducing any obvious changes in smooth muscle cells. After 4- to 10-min illumination, ICCs became progressively more damaged with swollen and ruptured mitochondria, loss of cytoplasmic contrast and detail, loss of caveolae, and rupture of the plasma membrane. No damage was seen in smooth muscle cells or nerves. Gap junctional ultrastructure was preserved. Intense illumination without preincubation with MB left the slow waves and the ultrastructure of ICC-CM preparations unaffected. In CM preparations, without the submuscular ICC-smooth-muscle network, MB plus light induced no changes in electrical activity. We conclude that the correlation between selective damage to the submuscular ICCs (relative to smooth muscle) and selective loss of the slow-wave activity (relative to other electrical activity of the CM) strongly indicates that the ICCs play an essential role in the generation of slow waves.

Animals↗

PubMed1993

Electrical coupling of circular muscle to longitudinal muscle and interstitial cells of Cajal in canine colon.

1. Electrical communication between circular muscle, longitudinal muscle and interstitial cells of Cajal (ICC) was investigated; the hypothesis was tested that the resting membrane potential (RMP) gradient in the circular muscle of canine colon is caused by electrical coupling to neighbouring cells. 2. Isolated longitudinal muscle exhibited spike-like action potentials at a RMP of -45 mV with a frequency and amplitude of 20 cycles/min and 12 mV, respectively. 3. The circular muscle (CM), devoid of longitudinal muscle, myenteric plexus and submuscular ICC-smooth-muscle network, was electrically quiescent at a uniform RMP of -62 mV across the entire circular muscle layer. 4. Preparations consisting of only the submuscular ICC network and a few adjacent layers of circular muscle cells exhibited slow wave-type action potentials at a RMP of about -80 mV. 5. In ICC-CM preparations, consisting of the submuscular ICC network and circular muscle, a RMP gradient of 10 mV was observed near the submucosal border, whereas the RMP was constant at -62 mV in the myenteric half of the circular muscle. 6. In full thickness (FT) preparations, a RMP gradient of 23 mV was observed. The RMP decreased gradually from -71 mV at the submucosal border to -48 mV at the myenteric border of the circular muscle. 7. Coupling of longitudinal muscle to circular muscle caused circular muscle cells at the myenteric surface to depolarize by 14 mV and longitudinal muscle cells to hyperpolarize by 3 mV. 8. In the ICC-CM preparations, the slow wave amplitudes did not decay exponentially away from the ICC network indicating that slow waves propagated actively into the circular muscle; in the FT preparations there was an apparent exponential decay but this was due to the RMP gradient. 9. Spike-like action potentials (SLAPs) superimposed on the plateau phase of slow waves did not decay exponentially away from the myenteric border suggesting that SLAPs were generated within the circular muscle layer. 10. In summary, circular muscle cells possess a uniform intrinsic RMP of -62 mV. The RMP gradient in situ is caused by electrical coupling of circular muscle cells to longitudinal muscle cells and the submuscular network of ICC. In situ, slow wave-type action potentials propagate actively into the circular muscle layer, and, dependent on the level of excitation, circular muscle cells actively generate spikes.

Action Potentials↗

PubMed1993

Selective accumulation of methylene blue by interstitial cells of Cajal in canine colon.

The network of interstitial cells of Cajal (ICC) at the submucosal surface of the canine colon was selectively stained by incubation with 15-50 microM methylene blue for 30-45 min. The network was composed of regularly scattered ICC cell bodies interconnected by long processes. Circular muscle cells were unstained. Staining of neurons was limited to one or two axons within bundles. The ICC network had a thickness of a single cell, since no overlapping of ICC cell bodies was observed. The ICC network connected the circular muscle cells at the submucosal surface across the septa which circumferentially divided the circular muscle into lamellae. Methylene blue at 50 microM slightly decreased the resting membrane potential and increased the duration of slow waves, leading to an increase in the force of phasic contractions, with no significant influence on other slow-wave parameters. Methylene blue produced neither electrophysiological nor mechanical effects on circular muscle preparations from which the submuscular ICC network was removed, indicating that the excitatory effects of methylene blue on the full-thickness circular muscle layer were mediated by ICC. In summary, the three-dimensional aspects of the submuscular ICC network can be visualized after selective staining by methylene blue. This staining does not affect physiological characteristics of smooth muscle cells.

Animals↗

PubMed1992

Intercellular communication in smooth muscle.

The functioning of a group of cells as a tissue depends on intercellular communication; an example is the spread of action potentials through intestinal tissue resulting in synchronized contraction. Recent evidence for cell heterogeneity within smooth muscle tissues has renewed research into cell coupling. Electrical coupling is essential for propagation of action potentials in gastrointestinal smooth muscle. Metabolic coupling may be involved in generation of pacemaker activity. This review deals with the role of cell coupling in tissue function and some of the issues discussed are the relationship between electrical synchronization and gap junctions, metabolic coupling, and the role of interstitial cells of Cajal in coupling.

Animals↗

PubMed1992

The fifth and sixth growth factor-like domains of thrombomodulin bind to the anion-binding exosite of thrombin and alter its specificity.

The domain of thrombomodulin that binds to the anion-binding exosite of thrombin was identified by comparing the binding of fragments of thrombomodulin to thrombin with that of Hirugen, a 12-residue peptide of hirudin that is known to bind to the anion-binding exosite of thrombin. Three soluble fragments of thrombomodulin, containing (i) the six repeated growth factor-like domains of thrombomodulin (GF1-6), (ii) one-half of the second through the sixth growth factor-like repeats (GF2.5-6), or (iii) the fifth and sixth such domains (GF5-6), were examined. Hirugen was a competitive inhibitor for either GF1-6 or GF2.5-6 stimulation of thrombin activation of protein C. GF5-6, which binds to thrombin without altering its ability to activate protein C, competed with fluorescein-labeled Hirugen for binding to thrombin. Therefore, all three thrombomodulin fragments, each of which lacked the chondroitin sulfate moiety, competed with Hirugen for binding to thrombin. To determine whether GF5-6 and Hirugen were binding to overlapping sites on thrombin or were interfering allosterically with each other's binding to thrombin, the effects of each thrombomodulin fragment and of Hirugen on the active site conformation of thrombin were compared using two different approaches: fluorescence-detected changes in the structure of the active site and the hydrolysis of chromogenic substrates. The GF5-6 and Hirugen peptides affected these measures of active site conformation very similarly, and hence GF5-6 and Hirugen contact residues on the surface of thrombin that allosterically alter the active site structure to a similar extent. Full-length thrombomodulin and GF1-6 alter the active site structure to comparable extents, but the amidolytic activity of thrombin complexed to thrombomodulin or GF1-6 differs significantly from that of thrombin complexed to GF5-6 or Hirugen. Taken together, these results indicate that the GF5-6 domain of thrombomodulin binds to the anion-binding exosite of thrombin. Furthermore, the binding of GF5-6 to the anion-binding exosite alters thrombin specificity, as evidenced by GF5-6-dependent changes in both the kcat and Km of synthetic substrate hydrolysis by thrombin. The contact sites on thrombin for the GF4 domain and the chondroitin sulfate moiety of thrombomodulin are still unknown.

Amino Acid Sequence↗

PubMed1992

Excitability of canine colon circular muscle disconnected from the network of interstitial cells of Cajal.

The 6 cpm omnipresent slow waves recorded in the circular muscle (CM) layer of canine colon are generated at the submucosal surface of the CM layer. After removal of the submucosal network of interstitial cells of Cajal (ICC), 66% of the CM preparations (25 of 38) were quiescent in Krebs solution. In the presence of carbachol, seven of nine of these spontaneously quiescent CM preparations demonstrated slow wave-like activity with mean frequency, duration and amplitude of 5.9 +/- 0.4 cpm, 2.8 +/- 0.5 s, and 0.8 +/- 0.2 mV, respectively. Similar slow wave-like activities were induced by TEA (seven out of eight quiescent CM preparations) with frequency, duration and amplitude of 6.1 +/- 0.2 cpm, 2.7 +/- 0.5 s, and 1.0 +/- 0.2 mV, respectively, and by BaCl2 (eight of eight quiescent CM preparations) with frequency, duration, and amplitude of 6.3 +/- 0.3 cpm, 1.8 +/- 0.2 s, and 0.5 +/- 0.1 mV, respectively. All the induced activities were abolished in the presence of 1 microM D600. CM preparations with the submucosal ICC network intact (ICC-CM) showed slow wave activity in Krebs solution at a frequency of 6.2 +/- 0.2 cpm, a duration of 3.6 +/- 0.2 s, and an amplitude of 1.0 +/- 0.1 mV (n = 22). When ICC-CM preparations were stimulated by BaCl2, carbachol, or TEA, the slow wave frequency did not change significantly, but the duration increased as well as the amplitude. In the presence of D600, the upstroke of slow waves remained and the frequency was not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Proteolytic formation of either of the two prothrombin activation intermediates results in formation of a hirugen-binding site.

Hirugen, a synthetic dodecapeptide corresponding to the carboxyl-terminal amino acids 53-64 of hirudin, binds within a deep groove in thrombin that contains a cationic region referred to as the anion-binding exosite. This region is important in many of the binary interactions of thrombin with macromolecular substrates and cofactors. Fluorescein-labeled hirugen was used to probe which steps in the prothrombin activation process generate this anion-binding exosite. Two activation cleavage sites exist in bovine prothrombin. Cleavage at Arg274-Thr275 releases the activation fragments to generate the thrombin precursor, prethrombin 2. Cleavage of prothrombin within a disulfide loop at Arg323-Ile324 leads to formation of meizothrombin with no loss of peptide material but with formation of amidolytic activity. Cleavage of the same bond in prethrombin 2 generates thrombin. Hirugen, labeled at the amino terminus with fluorescein isothiocyanate, does not bind to prothrombin but does bind to thrombin (Kd = 9.6 +/- 1.2 x 10(-8) M), prethrombin 2 (Kd = 1.3 +/- 0.1 x 10(-7) M), thrombin-fragment-2 complex (Kd = 1.1 +/- 0.2 x 10(-6) M), and meizothrombin (Kd = 1.6 +/- 0.5 x 10(-8) M). Prothrombin fragment-2 and hirugen both bind independently to thrombin. A ternary complex can form with hirugen and fragment-2 and either thrombin or prethrombin 2, suggesting that fragment-2 and hirugen bind to discrete sites. Hirugen also alters the active site conformation of thrombin as detected by modulation of synthetic substrate hydrolytic activity. These studies suggest that conformational changes, rather than alleviating steric hindrance, are responsible for the formation of the hirugen-binding site during prothrombin activation. Furthermore, this conformational change can be effected by the cleavage of either of the two bonds required for activation of prothrombin.

Amino Acid Sequence↗

PubMed1991

The region of the thrombin receptor resembling hirudin binds to thrombin and alters enzyme specificity.

A thrombin receptor has recently been cloned and the sequence deduced. The sequence reveals a thrombin cleavage site that accounts for receptor activation. The receptor also has an acidic region with some similarities to the carboxyl-terminal region of the leech thrombin inhibitor, hirudin. Synthetic peptides corresponding to the receptor cleavage site (residues 38-45), the hirudin-like domain (residues 52-69), and the covalently associated domains (residues 38-64) were evaluated for their ability to bind to thrombin. Peptides 38-45 and 38-64 were competitive inhibitors of thrombin's chromogenic substrate activity (Ki = 0.96 mM and 0.6 microM, respectively. Residues 52-69 altered the chromogenic substrate specificity, resulting in accelerated cleavage of some substrates and inhibited cleavage of others. The same peptide binds to thrombin and alters the fluorescence emission intensity of 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-thrombin in which the dansyl is attached directly to the active site serine (Kd = 32 +/- 7 microM). Residues 52-69 displace the carboxyl-terminal peptide of hirudin, indicating that they share a common binding site in the anion exosite of thrombin. These data suggest that the thrombin receptor has high affinity for thrombin due to the presence of the hirudin-like domain and that this domain alters the specificity of thrombin. This change in specificity may account for the ability of the receptor to serve as an excellent thrombin substrate despite the presence of an Asp residue in the P3 site, which is normally inhibitory to thrombin activity.

Amino Acid Sequence↗