Interlayer cation exchange stabilizes polar perovskite surfaces.

Global optimization is used to study the structure of the polar KTaO3 (001) surface. It is found that cation exchange near the surface leads to the most stable structure. This mechanism is likely to be general to metal oxides containing cations of differing charge.

Caveolin-1 inhibits matrix metalloproteinase-2 activity in the heart.

Apart from its ability to degrade extracellular matrix proteins, matrix metalloproteinase-2 (MMP-2) was recently revealed to have targets and actions within the cardiac myocyte. The localization of MMP-2 in caveolae of endothelial cells suggests that caveolin-1 (Cav-1) may play a role in regulating MMP-2. The caveolin scaffolding domain (CSD) of Cav-1 regulates several proteins including those involved with signaling cascades. Whether Cav-1 is responsible for regulating MMP-2 in the heart is unknown. Hearts from Cav-1(-/-) or Cav-1(+/+) mice were isolated and heart extracts or lipid raft enriched membrane fractions were prepared. MMP-2 activity in Cav-1(-/-) hearts was markedly enhanced when compared with Cav-1(+/+) hearts with no changes in MMP-2 protein levels between groups. In contrast, MMP-2 activity and protein level were greatly reduced in lipid raft enriched fractions of Cav-1(-/-) hearts. Purified CSD inhibited MMP-2 activity in a concentration-dependent manner as assessed using an in vitro degradation assay with a fluorogenic MMP-2 substrate (OmniMMP). These data suggest that Cav-1 plays a role in regulating MMP-2 activity. Cav-1 may thus be a novel mechanism to regulate MMP-2 activity in the heart.

Do gap junctions play a role in nerve transmissions as well as pacing in mouse intestine?

Varicosities of nitrergic and other nerves end on deep muscular plexus interstitial cells of Cajal or on CD34-positive, c-kit-negative fibroblast-like cells. Both cell types connect to outer circular muscle by gap junctions, which may transmit nerve messages to muscle. We tested the hypotheses that gap junctions transmit pacing messages from interstitial cells of Cajal of the myenteric plexus. Effects of inhibitors of gap junction conductance were studied on paced contractions and nerve transmissions in small segments of circular muscle of mouse intestine. Using electrical field stimulation parameters (50 V/cm, 5 pps, and 0.5 ms) which evoke near maximal responses to nitrergic, cholinergic, and apamin-sensitive nerve stimulation, we isolated inhibitory responses to nitrergic nerves, inhibitory responses to apamin-sensitive nerves and excitatory responses to cholinergic nerves. 18beta-Glycyrrhetinic acid (10, 30, and 100 microM), octanol (0.1, 0.3, and 1 mM) and gap peptides (300 microM of (40)Gap27, (43)Gap26, (37,43)Gap27) all failed to abolish neurotransmission. 18beta-Glycyrrhetinic acid inhibited frequencies of paced contractions, likely owing to inhibition of l-type Ca(2+) channels in smooth muscle, but octanol or gap peptides did not. 18beta-Glycyrrhetinic acid and octanol, but not gap peptides, reduced the amplitudes of spontaneous and nerve-induced contractions. These reductions paralleled reductions in contractions to exogenous carbachol. Additional experiments with gap peptides in both longitudinal and circular muscle segments after N(G)-nitro-l-arginine and TTX revealed no effects on pacing frequencies. We conclude that gap junction coupling may not be necessary for pacing or nerve transmission to the circular muscle of the mouse intestine.

Sodium-, chloride-, and mibefradil-sensitive calcium channels in intestinal pacing in wild-type and W/WV mice.

Pacing of intestinal smooth muscle is driven by a network of cells found in the myenteric plexus called the interstitial cells of Cajal (ICC-MP), which produce a rhythmic pacemaker current. Using intact segments of circular (CM) and longitudinal (LM) muscle from wild-type and W/WV mice, we found that sodium-, chloride-, and mibefradil-sensitive ion channel currents are required for normal pacing to occur. Application of 30 micromol/L and 300 micromol/L lidocaine, 1 mmol/L 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 50 nmol/L and 500 nmol/L mibefradil, or low sodium Krebs significantly reduced pacing frequency in LM and CM. However, simultaneously applying DIDS and lidocaine or low sodium Krebs solution did not completely block pacing nor did it have an additive effect. Lidocaine and low sodium Krebs solution also abolished the gradient of pacing frequencies (higher proximally) found throughout the intestine, resulting in a uniform contraction frequency of 30-40/min. In W/WV mice, which lack ICC-MP, application of DIDS and lidocaine had no effect on the robust pacing in LM segments. In conclusion we found that sodium-, chloride-, and mibefradil-sensitive channel activities were required for normal pacing and to maintain the pacing gradient found throughout the intestines in wild-type but not W/WV mice.

Caveolae and calcium handling, a review and a hypothesis.

Caveolae are associated with molecules crucial for calcium handling. This review considers the roles of caveolae in calcium handling for smooth muscle and interstitial cells of Cajal (ICC). Structural studies showed that the plasma membrane calcium pump (PMCA), a sodium-calcium exchanger (NCX1), and a myogenic nNOS appear to be colocalized with caveolin 1, the main constituent of these caveolae. Voltage dependent calcium channels (VDCC) are associated but not co-localized with caveolin 1, as are proteins of the peripheral sarcoplasmic reticulum (SR) such as calreticulin. Only the nNOS is absent from caveolin 1 knockout animals. Functional studies in calcium free media suggest that a source of calcium in tonic smooth muscles exists, partly sequestered from extracellular EGTA. This source supported sustained contractions to carbachol using VDCC and dependent on activity of the SERCA pump. This source is postulated to be caveolae, near peripheral SR. New evidence, presented here, suggests that a similar source exists in phasic smooth muscle of the intestine and its ICC. These results suggest that caveolae and peripheral SR are a functional unit recycling calcium through VDCC and controlling its local concentration. Calcium handling molecules associated with caveolae in smooth muscle and ICC were identified and their possible functions also reviewed.

Comparisons of neural and pacing activities in intestinal segments from W/W++ and W/W(V) mice.

We studied pacing and neurotransmission in longitudinal (LM) and circular muscle (CM) in intestine of W/W++ and W/W(V) mice. Electrical field-stimulation (EFS) of nerves in LM segments was more inhibitory in W/W(V) mice than in W/W++ mice. No inhibitory input to CM segments of W/W(V) mice was found. The EFS, after nerve block, entrained segments of both W/W++ and mutant mice with 10 ms pulses, and entrained those of mutant mice more readily at 1 and 3 ms pulses. Pacing with external electrodes did not depend on interstitial cells of Cajal in the myenteric plexus (ICC-MP). 2-Aminoethoxydiphenyl borate (2-APB), putative antagonist at IP3 receptors, store-operated channels and the Sacro-endoplasmic reticulum Ca2+ ATPase pump, reduced frequency and amplitudes of pacing of LM segments from W/W(V) mice as it did in BALB/c mice. Thus, its actions may not require ICC-MP. SKF 96365, a putative inhibitor of store-operated channels, reduced frequencies and amplitudes of intestinal segments in W/W++ mice at 10 or 30 micromol L-1. This resulted from blocking L-Ca2+-channels. Thus, no evidence was found that store-operated channels play a role in pacing. In LM segments of W/W(V), SKF 96365 had no effects on frequency of contractions. We conclude, results from models of severely reduced systems may not be applicable to intact ICC networks.

Role of glycosylation and membrane environment in nicotinic acetylcholine receptor stability.

The effects of glycosylation and membrane environment on the structural stability of the nicotinic acetylcholine receptor (nAChR) from Torpedo have been investigated to improve our understanding of factors that influence eukaryotic membrane protein crystallization. Gel shift assays and carbohydrate-specific staining show that the deglycosylation enzyme, Endo F1, removes at least 50% of membrane-reconstituted nAChR glycosylation. The extent of deglycosylation with Endo F1 increases upon detergent solubilization. Removal of between 60-100% of high mannose moieties from the nAChR has no effect on nAChR secondary structure, stability, or flexibility. Deglycosylation does not influence either agonist binding or the ability of the nAChR to undergo agonist-induced conformational change. In contrast, nAChR structural stability, flexibility, and function are all negatively influenced by simple changes in reconstituted membrane lipid composition. Our results suggest that deglycosylation may represent a feasible approach for enhancing the crystallizability of the nAChR. Our data also demonstrate that the dependence of nAChR structural stability on lipid environment may represent a significant obstacle to nAChR crystallization. Some membrane proteins may have evolved complex interactions with their lipid environments. Understanding the complexity of these interactions may be essential for devising an appropriate strategy for the crystallization of some membrane proteins.

Comparative study of knowledge about different Child Survival and Safe Motherhood intervention in two groups of mothers.

A cross-sectional study was conducted in an urban field practice slum area served by Urban Health Centre (UHC) attached to the Dept. of Preventive and Social Medicine, T. N. Medical College and Nair Hospital, Mumbai, to compare the knowledge about different Child Survival and Safe Motherhood interventions in two groups of mothers. 152 mother who regularly attended antenatal check-up in UHC constituted study group and 153 mothers selected by individual matching constituted the control group. Significant differences in the knowledge of study and control groups of mothers were observed about some interventions like time of initiation of breast feeding, duration of exclusive breast feeding, age of starting weaning and number of OPV and DPT doses to be given till 1 year of age.

Role of l-Ca(2+) channels in intestinal pacing in wild-type and W/W(V) mice.

Rhythmic contractions generating transit in the digestive tract are paced by a network of cells called interstitial cells of Cajal (ICC) found in the myenteric plexus (MP). ICC generate cyclic depolarizations termed "slow waves" that are passively transmitted to the smooth muscle to initiate contractions. The opening of l-Ca(2+) channels are believed to be primarily responsible for the influx of calcium generating a contraction in smooth muscle. However, l-Ca(2+) channels are not thought to be important in generating the pacing current found in ICC. Using intact segments of circular (CM) and longitudinal (LM) muscle from wild-type mice and mice lacking c-kit kinase (W/W(V)), we found that l-Ca(2+) channel currents are required for pacing at normal frequencies to occur. Application of 1 muM nicardipine caused a significant decrease in contraction amplitude and frequency in LM and CM that was successfully blocked with BAY K 8644. Nicardipine also abolished the pacing gradient found throughout the intestines, resulting in a uniform contraction frequency of 30-40/minute. Stimulating l-Ca(2+) channels with BAY K 8644 neither removed nor recovered the pacing gradient. W/W(V) mice, which lack ICC-MP, also exhibited a pacing gradient in LM. Application of nicardipine to LM segments of W/W(V) mouse intestine did not reduce pacing frequency, and in jejunum, resulted in a slight increase. BAY K 8644 did not affect pacing frequency in W/W(V) tissue. In conclusion, we found that l-Ca(2+) channel activity was required for normal pacing frequencies and to maintain the pacing frequency gradient found throughout the intestines in wild-type but not in W/W(V) mouse intestine.

Proteins of interstitial cells of Cajal and intestinal smooth muscle, colocalized with caveolin-1.

The murine jejunum and lower esophageal sphincter (LES) were examined to determine the locations of various signaling molecules and their colocalization with caveolin-1 and one another. Caveolin-1 was present in punctate sites of the plasma membranes (PM) of all smooth muscles and diffusely in all classes of interstitial cells of Cajal (ICC; identified by c-kit immunoreactivity), ICC-myenteric plexus (MP), ICC-deep muscular plexus (DMP), ICC-serosa (ICC-S), and ICC-intramuscularis (IM). In general, all ICC also contained the L-type Ca(2+) (L-Ca(2+)) channel, the PM Ca(2+) pump, and the Na(+)/Ca(2+) exchanger-1 localized with caveolin-1. ICC in various sites also contained Ca(2+)-sequestering molecules such as calreticulin and calsequestrin. Calreticulin was present also in smooth muscle, frequently in the cytosol, whereas calsequestrin was present in skeletal muscle of the esophagus. Gap junction proteins connexin-43 and -40 were present in circular muscle of jejunum but not in longitudinal muscle or in LES. In some cases, these proteins were associated with ICC-DMP. The large-conductance Ca(2+)-activated K(+) channel was present in smooth muscle and skeletal muscle of esophagus and some ICC but was not colocalized with caveolin-1. These findings suggest that all ICC have several Ca(2+)-handling and -sequestering molecules, although the functions of only the L-Ca(2+) channel are currently known. They also suggest that gap junction proteins are located at sites where ultrastructural gap junctions are know to exist in circular muscle of intestine but not in other smooth muscles. These findings also point to the need to evaluate the function of Ca(2+) sequestration in ICC.

Communication between interstitial cells of Cajal and gastrointestinal muscle.

Interstitial cells of Cajal (ICC) pace gastrointestinal muscle by initiating slow waves in both muscle layers and appear to be preferred sites for reception of neurotransmitters. ICC of the myenteric plexus (ICC-MP) pace stomach and small intestine, while intramuscular ICC (ICC-IM) receive nerve messages. Recently, ICC-IM have been found to provide regenerative responses to and amplification of pacing messages from ICC-MP, at least in some systems. This review will examine the assumption that gap junctions provide low-resistance contacts for pacing. Structural and functional evidence will be evaluated. Structural, theoretical and experimental difficulties with the gap junctions hypothesis for pacing will be considered. So far little direct evidence about the role of gap junctions in neurotransmission exists, although a structural basis exists. Alternate possibilities for transmission of ICC pacing and neural messages will be examined and suggestions for future research made.

Changes in membrane cholesterol affect caveolin-1 localization and ICC-pacing in mouse jejunum.

Pacing of mouse is dependent on the spontaneous activity of interstitial cells of Cajal in the myenteric plexus (ICC-MP). These ICC, as well as intestinal smooth muscle, contain small membrane invaginations called caveolae. Caveolae are signaling centers formed by insertions of caveolin proteins in the inner aspect of the plasma membrane. Caveolins bind signaling proteins and thereby negatively modulate their signaling. We disrupted caveolae by treating intestinal segments with methyl beta-clodextrin (CD) to remove cholesterol or with water-soluble cholesterol (WSC) to load cholesterol. Both of these treatments reduced pacing frequencies, and these effects were reversed by the other agent. These treatments also inhibited paced contractions, but complete reversal was not observed. To evaluate the specificity of the effects of CD and WSC, additional studies were made of their effects on responses to carbamoyl choline and to stimulation of cholinergic nerves. Neither of these treatments affected these sets of responses compared with their respective time controls. Immunochemical and ultrastructural studies showed that caveolin 1 was present in smooth muscle membranes and ICC-MP. CD depleted both caveolin 1 and caveolae, whereas WSC increased the amount of caveolin 1 immunoreactivity and altered its distribution but failed to increase the number of caveolae. The effects of each agent were reversed in major part by the other. We conclude that signaling through caveolae may play a role in pacing by ICC but does not affect responses to acetylcholine from nerves or when added exogenously.

ICC pacing mechanisms in intact mouse intestine differ from those in cultured or dissected intestine.

Pacing of mouse intestine is driven by spontaneous activity of a network of interstitial cells of Cajal in the myenteric plexus (ICC-MP). So far, highly dissected circular muscle (CM) strips from control and mutant mice lacking ICC-MP and isolated, cultured ICC from newborn control mice were used to analyze its properties. Using intact circular and longitudinal segments of intestine, we recently reported that there were both significant similarities and differences between pacing studied in segments and from isolated, dissected tissues. Here, we report additional similarities and differences in our model from those in highly reduced systems. Similar to cultured or dissected intestine, blockade of sarcoplasmic-endoplasmic reticulum Ca(2+) pumps with thapsigargin or cyclopiazonic acid reduced pacing frequency, but thapsigargin was less effective than in isolated, cultured ICC. Moreover, inhibition of inositol 1,4,5-trisphosphate (IP(3)) receptors with xestospongin C, a putative inhibitor of IP(3) receptors, failed to affect pacing but successfully blocked increased pacing frequency by phorbol ester. 2-Aminoethoxy-diphenylborate, a putative blocker of IP(3)-mediated calcium release, caused a significant decrease in the amplitude and frequency of contractions. The mitochondrial uncoupler carbonyl cyanide p-trifluormethoxyphenylhydrazone blocked pacing and KCl-induced contractions at a concentration of 1 microM. The cyclic nucleotide agonists sodium nitroprusside (SNP), forskolin, and 8-bromo-cGMP inhibited pacing in CM. In longitudinal muscle (LM), SNP and forskolin had little effect on pacing. Furthermore, dibutyryl-cAMP did not affect pacing in CM or LM. These results suggest that pacing in intact intestine is under partly similar regulatory control as in more reduced systems. However, pacing in intact intestine is not affected by xestospongin C, which abolishes pacing in isolated, cultured ICC and exhibits attenuated responses to thapsigargin. Also, major differences between LM and CM suggest a separate pacemaker may drive LM.

A new model of pacing in the mouse intestine.

A simple model of pacing in mouse intestine to longitudinal (LM) as well as circular muscle (CM) has been developed. Undissected segments of LM or CM from mouse ileum or jejunum were prepared to record contractions, nerve functions were inhibited, and regular spontaneous contractions were recorded. These had the properties expected of interstitial cells of Cajal (ICC) paced contractions: ileum slower than jejunum, inhibited but not abolished by nicardipine, reduced in frequency by cyclopiazonic acid, abolished by Ca(2+)-free media, and high temperature dependence (Q10 approximately 2.6-3.2). Nicardipine significantly reduced the pacing frequency in LM and CM. Intestinal segments from W/W(V) mice had few irregular contractions in CM but had regular contractions in LM. Other differences were found between LM and CM that suggest that the control of pacing of LM differed from pacing of CM. Moreover, both LM and CM segments in wild-type and W/W(V) and after cyclopiazonic acid responded to electrical pacing (50 V/cm, 50 or 100 ms) at 1 pulse per second. Temperature <26 degrees C inhibited electrically paced contractions in CM. These findings suggest that the current models of ICC pacing need to be modified to apply to intact segments of mouse intestine.

Does ICC pacing require functional gap junctions between ICC and smooth muscle in mouse intestine?

We tested the hypothesis that interstitial cells of Cajal (ICC) pace longitudinal and circular muscle of mouse intestine through gap junctions. Carbenoxolone (10(-6), 10(-5), 10(-4) mol L(-1)), an inhibitor of gap junction conductance, was applied to segments of longitudinal or circular muscle with contractions driven by ICC after inhibition of nerve function by tetrodotoxin (10(-6) mol L(-1)) and L-NOARG (10(-4) mol L(-1)). Carbenoxolone concentration- and time-dependently inhibited the amplitude of contraction (0.2-1.5 g in controls) of segments of longitudinal muscle, but had no effect on the frequency of contractions (from 36-54 min). It also inhibited the amplitude of contractions of circular muscle segments and reduced the frequency slightly at 10(-4) mol L(-)1. Carbenoxolone inhibited tonic contractions of longitudinal but not circular segments to 60 mmol L(-1) KCl, suggesting that it directly inhibited contractions of longitudinal muscle. The responses to pacing by electrical field stimulation (40 V cm(-1), 50-100 ms, 1 Hz) after block of nerve function were reduced insignificantly in amplitude, and not in frequency in both longitudinal and circular segments. We conclude that it is likely that only gap junctions within circular muscle are involved in pacing of muscle by ICC. Carbenoxolone also has effects on muscle contractility in longitudinal muscle.

Local, exendin-(9-39)-insensitive, site of action of GLP-1 in canine ileum.

Glucagon-like peptide-1 (GLP-1) modulates glucose levels following a meal, including by inhibition of gastric emptying and intestinal transport. Intra-arterial injection of GLP-1 into the gastric corpus, antrum, or pylorus of anesthetized dogs had no effect on the contractile activity of the resting or neurally activated stomach. GLP-1 injected intra-arterially inhibited intestinal segments when activated by enteric nerve stimulation but not by acetylcholine. Isolated ileum segments were perfused intra-arterially, instrumented with strain gauges to record circular muscle activity and with subserosal electrodes to stimulate enteric nerves. GLP-1 caused concentration-dependent inhibition of nerve-stimulated phasic but not tonic activity. This was absent during TTX-induced activity and partly prevented by N(G)-nitro-L-arginine. Exendin-(9-39), the GLP-1 antagonist, had no intrinsic activity and did not affect the actions of GLP-1. Capsaicin mimicked the effects of GLP-1 and may have reduced the effect of subsequent GLP-1. GLP-1 may mediate paracrine action on afferent nerves in the canine ileal mucosa using an unusual receptor.

Evidence for the existence of a constitutive nitric oxide synthase in vascular smooth muscle.

1. We have identified a neuronal nitric oxide synthase (NOS)-like constitutive form of NOS in vascular smooth muscle (VSM) using a functional contractility approach as well as immunohistochemical methods. 2. N(G)-Nitro-L-arginine methyl ester, N(G)-monomethyl-L- arginine and N(G)-nitro-L-arginine (L-NOARG), the competitive inhibitors of NOS, inhibited Mg(2+)-induced relaxation of de-endothelialized rat aorta precontracted with phenylephrine (PE). This Mg(2+) relaxation of VSM was not affected by inhibitors of inducible NOS. 3. Electrical field stimulation (EFS; 30-70 Hz) caused relaxation of rat aorta in the presence of tetrodotoxin (therefore not a neurogenic effect) and this EFS relaxation was effectively inhibited by L-NOARG, oxyhemoglobin and methylene blue. 4. Immunohistochemical studies of dog saphenous vein using antibodies raised against neuronal NOS indicated prominent staining along the plasmalemma in a punctate pattern similar to the distribution of antibodies against caveolin-1, a major constituent of the plasmalemmal caveolae. 5. We propose that a constitutive NOS of non-endothelial, non-neuronal origin is present in a special caveolae domain of VSM cell membranes and could be activated by an ionic mechanism yet to be characterized.

Roles of guanylate cyclase in responses to myogenic and neural nitric oxide in canine lower esophageal sphincter.

Whether cGMP and cytosolic guanylate cyclase (cGC) mediate responses of canine lower esophageal sphincter (LES) to nitric oxide (NO) released from nerves, produced in muscle, or added exogenously was evaluated in vitro. 1-H-(1,2,4)oxadiazole(4,3-alpha)quinoxalin-1-1 (ODQ), inhibitor of cGC, reduced relaxations to nerve stimulation and sodium nitroprusside but not to nitric-oxide synthase activity-dependent outward K(+)-currents in isolated muscle cells. ODQ also failed to increase tone after nerve blockade. Nonspecific K(+) channel blocker, TEA ion at 20 mM was previously shown to increase tone, occlude NO-mediated modulation of tone, and inhibit NO-dependent outward currents but not neural relaxation in LES cells. In this study, TEA abolished neural relaxation and nearly abolished relaxation to sodium nitroprusside when present with ODQ. We conclude that mechanisms coupling NO in canine LES to responses vary with the source of NO. ODQ-dependent mechanisms, presumably involving cGC, mediate actions of NO from nerves, but NO from muscle utilizes TEA-sensitive but not ODQ-dependent mechanisms to modulate tone and outward currents. Exogenous NO utilizes both TEA- and ODQ-dependent mechanisms.

Physiology and pathophysiology of the interstitial cell of Cajal: from bench to bedside. III. Interaction of interstitial cells of Cajal with neuromediators: an interim assessment.

Interstitial cells of Cajal (ICC) control gastrointestinal motility; some pace slow waves and others act in enteric neurotransmission. This review asks the question, does either class of ICC receive and respond to messages carried by neuromediators from these nerves? Relevant evidence includes the presence of receptors or responses to exogenous neuromediators and responses to endogenous neuromediators. Some pacemaking ICC networks have receptors for or respond to some exogenous neuromediators. None is known to respond to endogenous neuromediators. Intramuscular ICC have receptors for and respond to some neuromediators and are required in mice for responses to the exogenous and endogenous neuromediators nitric oxide and acetylcholine. The mechanisms underlying this requirement remain unclear. ICC pathologies exist, but their origins are unknown.