Effects of haloperidol, clotrimazole, and pyridoxal-5′-phosphate on synaptic transmission in smooth muscles of the human colon

In strips of smooth muscles of the human colon, haloperidol (Hal) and clotrimazole (Clo), in contrast to pyridoxal-5′-phosphate (PP), suppressed spontaneous electrical and contractile activities of these strips and also post-inhibitory excitation developing after inhibitory synaptic potentials (ISPs). Haloperidol, Clo, PP, and PP applied against the background of the action of Nω-nitro-L-arginine noticeably changed the parameters of ISPs. The pattern of effect of Hal on synaptic inhibition in smooth muscles was preserved against the background of the action of PP, and that of PP was preserved against the background of the action of Hal. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 408–411, July–October, 2007.     

Hyperpolarization contributes to vascular hyporeactivity in rats with lipopolysaccharide-induced endotoxic shock

We have examined the role of membrane hyperpolarization in mediating vascular hyporeactivity induced by bacterial lipopolysaccharide (LPS) in endothelial-denuded strips of rat thoracic aorta ex vivo. The injection of rats with LPS caused a significant fall of blood pressure and a severe vascular hyporeactivity to norepinephrine. The membrane potential recording showed that endotoxemia caused a hyperpolarization when compared to the control. This hyperpolarization was fully restored by methylene blue (MB; 10 microM) and partially reversed by Nomega-nitro-L-arginine methyl ester (L-NAME; 0.3 mM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 microM), tetraethylammonium (TEA; 10 mM), charybdotoxin (CTX; 0.1 microM), or glibenclamide (GB; 10 microM), however, this hyperpolarization was not significantly affected by apamin (0.1 microM), 4-aminopyridine (4-AP; 1 mM), or Ba2+ (50 microM). In addition, the basal tension of the tissues obtained from endotoxemic rats was enhanced by the following order: MB > or = ODQ > TEA > or = L-NAME > or = CTX > GB; whereas apamin, 4-AP or Ba2+ had no significant effects on these tissues. In contrast, none of these inhibitors had significant effects on the membrane potential or the basal tension in control tissues. Our electrophysiological results further confirmed previous studies showing that in addition to nitric oxide, the large conductance Ca2+-activated K+-channels and ATP-sensitive K+-channels are, most likely, responsible for endotoxin-mediated hyporeactivity to vasoconstrictor agents in vascular smooth muscle.     

An influence of ligands of metabotropic glutamate receptor subtypes on parkinsonian-like symptoms and the striatopallidal pathway in rats

Summary. Several data indicate that inhibition of glutamatergic transmission may be important to alleviate of parkinsonian symptoms. Therefore, the aim of the present paper is to review recent studies on the search for putative antiparkinsonian-like effects of mGluR ligands and their brain targets. In order to inhibit glutamatergic transmission, the group I mGluRs (mGluR1 and mGluR5) were blocked, and group II (mGluR2/3) or III (mGluR4/7/8) mGluRs were activated. Systemic or intrastriatal administration of group I mGluR antagonists (mGluR5 – MPEP, MTEP; mGluR1 – AIDA) was found to inhibit parkinsonian-like symptoms (catalepsy, muscle rigidity) in rats. MPEP administered systemically and mGluR1 antagonists (AIDA, CPCCOEt, LY367385) injected intrastriatally reversed also the haloperidol-increased proenkephalin (PENK) mRNA expression in the striatopallidal pathway. Similarly, ACPT-1, a group III mGluR agonist, administered into the striatum, globus pallidus or substantia nigra inhibited the catalepsy. Intrastriatal injection of this compound reduced the striatal PENK expression induced by haloperidol. In contrast, a group II mGluR agonist (2R,4R-APDC) administered intrastriatally reduced neither PENK expression nor the above-mentioned parkinsonian-like symptoms. Moreover, a mixed mGluR8 agonist/AMPA antagonist, (R,S)-3,4-DCPG, administered systemically evoked catalepsy and enhanced both the catalepsy and PENK expression induced by haloperidol. The results reviewed in this article seem to indicate that group I mGluR antagonists or some agonists of group III may possess antiparkinsonian properties, and point at the striatopallidal pathway as a potential target of therapeutic intervention.     

Morphogenesis and regeneration from stomatal guard cell complexes of cotton (Gossypium hirsutum L.)

 The development of a regeneration system from cotton stomatal guard cells directly on epidermal strips is described. The most important factors affecting embryogenic callus initiation in both of the varieties tested (Coker 312 and 315) were the source of the epidermal tissue, including plant age (4–5 months old), the developmental stage of the flower (opening flower stage) from which bracts were obtained, the composition of the culture medium and light irradiance. The flower developmental stage was critical for callus formation, which was observed only from bracts obtained from opening flowers. In addition, epidermal strips excised from the bract basal region were more responsive in culture than those obtained from the top region. Improved callus initiation was obtained on epidermal strips which had their cuticle in contact with the culture medium. Light irradiance was a limiting factor for embryogenic callus formation, which was observed only in calluses cultured under the lower light irradiance (15.8 μmol m^–2 s^–1). Somatic embryogenesis was observed on callus cultures subcultured consecutively to a culture medium containing naphthalene acetic acid (10.7 μM) and isopentenyladenine (4.9 μM). Histodifferentiation of somatic embryos was improved on a medium containing naphthaleneacetic acid (8.1 μM)+isopentenyladenine (2.5 μM) and abscisic acid (0.19–0.38 μM). Somatic embryo germination and plantlet development were obtained using established protocols with few modifications. On average, one fully developed plant was obtained from the culture of circa 100 epidermal strips in both cultivars. Received: 19 May 2000 / Revision received: 25 August 2000 / Accepted: 29 August 2000     

Periphyton biomass,potential production and respiration in a shallow lake during winter and spring

Abundance, depth distribution, potential productivity and respiration of periphyton on short-time (1 month) and long-time incubated strips were followed monthly during the winter–spring (January–May) transition in a shallow eutrophic lake. A taxonomic shift occurred from dominance of diatoms under ice to chlorophyte dominance in spring communities on the long-time incubated strips, while diatoms dominated until May on the short-time incubated strips. Periphyton biomass accrual was low during the ice-covered winter months (November–January: 4 mg chl a m⁻² month⁻¹), but increased to a maximum of 112 mg chl a m⁻² month⁻¹ immediately after ice-out in February. During February–April, the biomass remained constant before declining in May. Periphyton on long-time incubated strips was equally distributed in the water column in winter (January–February), but was higher near the water surface in spring (March–May). Periphyton did not change with depth on the short-time incubated strips. The potential production to respiration ratio (P/R) was negatively correlated with periphyton biomass. Throughout the study, P/R was <1 for the short-time incubated periphyton, while this was only the case in March–April for the long-time incubations. This study showed a high productive capacity of winter periphyton, resulting in accumulation of a relatively high periphytic biomass early in the season. A massive periphyton density in eutrophic lakes already in winter–spring may potentially delay or prevent the establishment and re-occurrence of submerged macrophytes in the early oligotrophication phase following a reduction of the external nutrient loading. Handling editor: Luigi Naselli-Flores     

Upregulation of RGS4 and downregulation of CPI-17 mediate inhibition of colonic muscle contraction by interleukin-1

The pro-inflammatory cytokine IL-1β contributes to the reduced contractile responses of gut smooth muscle observed in both animal colitis models and human inflammatory bowel diseases. However, the mechanisms are not well understood. The effects of IL-1β on the signaling targets mediating acetylcholine (ACh)-induced initial and sustained contraction were examined using rabbit colonic circular muscle strips and cultured muscle cells. The contraction was assessed through cell length decrease, myosin light chain (MLC₂₀) phosphorylation, and activation of PLC-β and Rho kinase. Expression levels of the signaling targets were determined by Western blot analysis and real-time RT-PCR. Short interfering RNAs (siRNAs) for regulator of G protein signaling 4 (RGS4) were used to silence endogenous RGS4 in muscle strips or cultured muscle cells. IL-1β treatment of muscle strips inhibited both initial and sustained contraction and MLC₂₀ phosphorylation in isolated muscle cells. IL-1β treatment increased RGS4 expression but had no effect on muscarinic receptor binding or G_q expression. In contrast, IL-1β decreased the expression and phosphorylation of CPI-17 but had no effect on RhoA expression or ACh-induced Rho kinase activity. Upregulation of RGS4 and downregulation of CPI-17 by IL-1β in muscle strips were corroborated in cultured muscle cells. Knockdown of RGS4 by siRNA in both muscle strips and cultured muscle cells blocked the inhibitory effect of IL-1β on initial contraction and PLC-β activation, whereas overexpression of RGS4 inhibited PLC-β activation. These data suggest that IL-1β upregulates RGS4 expression, resulting in the inhibition of initial contraction and downregulation of CPI-17 expression during sustained contraction in colonic smooth muscle. rabbit; short interfering RNA; acetylcholine; phospholipase C-β; Rho kinase; regulator of G protein signaling 4     

Small-conductance, Ca(2+) -activated K+ channel 2 is the key functional component of SK channels in mouse urinary bladder

Small-conductance Ca(2+)-activated K(+) (SK) channels play an important role in regulating the frequency and in shaping urinary bladder smooth muscle (UBSM) action potentials, thereby modulating contractility. Here we investigated a role for the SK2 member of the SK family (SK1-3) utilizing: 1) mice expressing beta-galactosidase (beta-gal) under the direction of the SK2 promoter (SK2 beta-gal mice) to localize SK2 expression and 2) mice lacking SK2 gene expression (SK2(-/-) mice) to assess SK2 function. In SK2 beta-gal mice, UBSM staining was observed, but staining was undetected in the urothelium. Consistent with this, urothelial SK2 mRNA was determined to be 4% of that in UBSM. Spontaneous phasic contractions in wild-type (SK2(+/+)) UBSM strips were potentiated (259% of control) by the selective SK channel blocker apamin (EC(50) = 0.16 nM), whereas phasic contractions of SK2(-/-) strips were unaffected. Nerve-mediated contractions of SK2(+/+) UBSM strips were also increased by apamin, an effect absent in SK2(-/-) strips. Apamin increased the sensitivity of SK2(+/+) UBSM strips to electrical field stimulation, since pretreatment with apamin decreased the frequency required to reach a 50% maximal contraction (vehicle, 21 +/- 4 Hz, n = 6; apamin, 12 +/- 2 Hz, n = 7; P < 0.05). In contrast, the sensitivity of SK2(-/-) UBSM strips was unaffected by apamin. Here we provide novel insight into the molecular basis of SK channels in the urinary bladder, demonstrating that the SK2 gene is expressed in the bladder and that it is essential for the ability of SK channels to regulate UBSM contractility.     

Adventitious shoot production from strips of stem in the Dutch elm hybrid ‘Commelin’: plantlet regeneration and neomycin sensitivity

The regenerative ability of small strips of stem of the Dutch elm hybrid ‘Commelin’ was tested as well as its sensitivity to neomycins. Cambium explants (1 mm thick), were excised from woody stems collected in the field. Up to 20 buds/explant were induced within 2–3 weeks giving 2–5 rootable shoots/explant after 5–6 weeks. Shoot excision every week from week three improved the yield up to 7 shoots per explant. Fourteen and 2.9 μM GA3 promoted shoot growth. Cytokinins (1 μM zeatin or 5 μM BA or 0.05 μM TDZ) completely inhibited shoot production and promoted callus formation. Kanamycin and paromomycin at between 240 and 360 μM inhibited shoot formation as did geneticin at 80 μM. The shoot-forming ability of the explants was high from leaf fall in the autumn to the spring flush, but could be maintained up to September by using cold storage (5–7 °C). Ninety-six percent of the shoots rooted with 0.5 μM IBA and were successfully acclimatized despite having a large basal callus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Time course of neural and contractile disturbances in a rat model of colitis induced by Trichinella spiralis

Colitis induced by Trichinella spiralis in rat induces alterations in the spontaneous motor pattern displayed by circular colonic muscle [Auli, M., Fernandez, E., 2005. Characterization of functional and morphological changes in a rat model of colitis induced by T. spiralis. Digestive Diseases and Sciences 50(8), 1432-1443]. We examined the temporal relationship between the severity of inflammation and the altered contractility of the underlying circular muscle as well as the role of NANC inhibitory pathways in the disruption of the motility pattern. Colitis was induced by intrarectal administration of T. spiralis larvae. Responses to acetylcholine (ACh) and increased extracellular potassium as well as the effect of tetrodotoxin (TTX, 1 microM), N-nitro-l-arginine (L-NOARG, 1 mM) and apamin (1 microM) were determined in vitro in the organ bath with circular muscle strips from sham-infected and infected rats at days 2-30 postinfection (PI). Microelectrode recordings were performed to study the putative changes in electrical activity of colonic smooth muscle cells. Responses to ACh and KCl were decreased at all days PI compared to sham. Intracellular calcium depletion had a greater inhibitory effect in inflamed tissue (6-14 PI). The effect of TTX, L-NOARG and apamin on the spontaneous contractions was found to be altered in all infected rats, i.e. their effects were transient and milder. Inflamed tissue showed lower resting membrane potential and a decreased duration of inhibitory junction potentials induced by electrical stimulation. These data suggest that the decreased contractility of colonic circular smooth muscle induced by the intrarectal T. spiralis infection results from the impairment of the excitation-contraction coupling, from a persistent hyperpolarization of smooth muscle cells and from impaired NANC inhibitory neurotransmission.     

Chlorophyll is not the primary photoreceptor for the stimulation of P-type H+ pump and growth in variegated leaves of Coleus×hybridus

There has been persisting controversy over the role of photosynthesis in the stimulation of the plasma membrane H⁺-ATPase and growth of dicotyledonous leaves by light. To investigate this, we compared the effects of light on growth, H⁺ net efflux and membrane potential (V_m) of strips which contained either only chlorophyll-free (white) mesophyll cells or chlorophyll-containing (green) cells cut from variegated Coleus leaves. White mesophyll cells responded to white, blue and red light with a hyperpolarization of V_m, an acidification of the apoplast and a promotion of growth, all of which began after a lag of 2–7 min. In contrast, green mesophyll cells showed a biphasic light response in which the hyperpolarization and the acidification were preceded by a rapid depolarization of V_m and an alkalinization of the apoplast. Nevertheless, green and white tissues showed comparable growth promotions in response to light. The light response of the leaf mesophyll is a composite of two separate photosystems. The initial depolarization and alkalinization are mediated by photosynthesis and blocked by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The slower hyperpolarization, acidification and growth response, on the other hand, are clearly in response to light absorption by pigments other than chlorophyll. Received: 11 February 2000 / Accepted: 2 May 2000     

Locomotor reactions and excitability of neurons during the blockade of dopamine by haloperidol in vertebrate and invertebrate animals

Two groups of rats with different level of motor activities: high- and low-active animals, were distinguished. The blockade of dopamine receptors by haloperidol led to depression of locomotor activity in both groups of rats; in grape snails, haloperidol caused a decrease of the velocity of locomotor responses. In was found that within 5 minutes of intravenous injection of haloperidol the excitability of spinal centers of rats decreased; but in 30 minutes in started restoring. Chronic application of the preparation depressed the effect of posttetanic potentiation of H-response in gastrocnemius muscle of spinal rats. In command neurons of grape snail, chronic injections of haloperidol causes a significant hyperpolarization shift of membrane potential and an increase of threshold of the generation of action potential. It was shown that the selective pharmacological inhibition of dopaminergic system of the brain led to a decrease of excitability in some determined neurons of the snail and spinal motor centers of rats, as well as inhibited the locomotor responses both in vertebrate and in invertebrate animals.     

Apamin inhibits NO-induced relaxation of the spontaneous contractile activity of the myometrium from non-pregnant women

There is now considerable evidence for the involvement of K⁺ channels in nitric oxide (NO) induced relaxation of smooth muscles including the myometrium. In order to assess whether apamin-sensitive K⁺ channels play a role in NO – induced relaxation of the human uterus, we have studied the effect of specific blockers of these channels on the relaxation of myometrium from non-pregnant women. In vitro isometric contractions were recorded in uterine tissues from non-pregnant premenopausal women who had undergone hysterectomy. Apamin (10 nM) and scyllatoxin (10 nM) did not alter spontaneous myometrial contractions. However, 15-min pretreatment of the myometrium strips with apamin completely inhibited relaxation caused by diethylamine-nitric oxide (DEA/NO). The pretreatment with scyllatoxin significantly reduced (about 2.6 times) maximum relaxation of the strips induced by DEA/NO (p < 0.05). These results strongly suggest that, beside Ca²⁺ and voltage dependent charybdotoxin-sensitive (CTX-sensitive) K⁺ channels, apamin-sensitive K⁺ channels are also present in the human non-pregnant myometrium. These channels offer an additional target in the development of new tocolytic agents.     

Small- and intermediate-conductance Ca2+-activated K+ channels directly control agonist-evoked nitric oxide synthesis in human vascular endothelial cells

The contribution of small-conductance (SK_Ca) and intermediate-conductance Ca²⁺-activated K⁺ (IK_Ca) channels to the generation of nitric oxide (NO) by Ca²⁺-mobilizing stimuli was investigated in human umbilical vein endothelial cells (HUVECs) by combining single-cell microfluorimetry with perforated patch-clamp recordings to monitor agonist-evoked NO synthesis, cytosolic Ca²⁺ transients, and membrane hyperpolarization in real time. ATP or histamine evoked reproducible elevations in NO synthesis and cytosolic Ca²⁺, as judged by 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) and fluo-3 fluorescence, respectively, that were tightly associated with membrane hyperpolarizations. Whereas evoked NO synthesis was unaffected by either tetraethylammonium (10 mmol/l) or BaCl₂ (50 µmol/l) + ouabain (100 µmol/l), depleting intracellular Ca²⁺ stores by thapsigargin or removing external Ca²⁺ inhibited NO production, as did exposure to high (80 mmol/l) external KCl. Importantly, apamin and charybdotoxin (ChTx)/ triarylmethane (TRAM)-34, selective blockers SK_Ca and IK_Ca channels, respectively, abolished both stimulated NO synthesis and membrane hyperpolarization and decreased evoked Ca²⁺ transients. Apamin and TRAM-34 also inhibited an agonist-induced outwardly rectifying current characteristic of SK_Ca and IK_Ca channels. Under voltage-clamp control, we further observed that the magnitude of agonist-induced NO production varied directly with the degree of membrane hyperpolarization. Mechanistically, our data indicate that SK_Ca and IK_Ca channel-mediated hyperpolarization represents a critical early event in agonist-evoked NO production by regulating the influx of Ca²⁺ responsible for endothelial NO synthase activation. Moreover, it appears that the primary role of agonist-induced release of intracellular Ca²⁺ stores is to trigger the opening of both K_Ca channels along with Ca²⁺ entry channels at the plasma membrane. Finally, the observed inhibition of stimulated NO synthesis by apamin and ChTx/TRAM-34 demonstrates that SK_Ca and IK_Ca channels are essential for NO-mediated vasorelaxation. calcium; endothelium; hyperpolarization; small-conductance calcium-activated potassium channel; intermediate-conductance calcium-activated potassium channel channel     

Inhibitory and excitatory mechanisms of neurotensin action in canine intestinal circular muscle in vitro.

The effect of neurotensin on canine ileal circular muscle devoid of myenteric plexus was investigated using single and double sucrose gap techniques. Similar results were obtained with microelectrode techniques. Neurotensin caused a temperature-sensitive and dose-dependent biphasic response, an initial hyperpolarization associated with inhibition of contractile activity, followed by an excitatory phase, usually consisting of spike discharge and tonic and phasic contractions, for which depolarization was not required. Neither response was affected by tetrodotoxin, phentolamine, propranolol, or atropine. The hyperpolarization was associated with decreased membrane resistance, blocked by 10(-7) M apamin, and converted to tonic depolarization by apamin (10(-6) M). Tachyphylaxis to neurotensin occurred when the stimulation interval was less than 20 min. After Ca2+ depletion, depolarization was observed instead of the hyperpolarization; this depolarization was not affected by nitrendipine and was gradually abolished with repetitive stimulation at 20-min intervals. When Ca2+ was present, nifedipine did not alter the hyperpolarizing phase of the response but inhibited spiking and blocked all contractions. The excitatory phase of the response was enhanced by Bay K-8644. Neuromedin N elicited a response identical with that of neurotensin. The responses of the two peptides were completely cross tachyphylactic. Inhibitory junction potentials were not affected by neurotensin tachyphylaxis. It is concluded that neurotensin and neuromedin N activate apamin-sensitive, calcium-dependent potassium channels in circular muscle, causing membrane hyperpolarization and inhibition of muscle contraction. Release of intracellular calcium is involved in the activation of these potassium channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelin-1 induced sustained contractions of tracheal smooth muscle involve an activation of protein kinase C

Endothelin-1, a potent vasoconstrictor peptide produces concentration dependent contractions in lamb tracheal smooth muscle. These contractions are not inhibited by low doses (up to 20 μM) of trifluoroperazine and W-7, the calmodulin/myosin light chain kinase (MLCK) inhibitors. At higher concentrations (200 μM), a delayed and poor reversal of isometric tensions results. These relaxations are coupled with a partial dephosphorylation of regulatory myosin light chain (MLC). Preincubation of fiber strips in MLCK inhibitors (200 μM) results in a delayed and attenuated contractile response but without a dephosphorylation of MLC. H-7, a putative protein kinase C antagonist (25–100 μM) abolishes endothelin-1 induced contractile effects rapidly (50% relaxation within 1–3 min). Moreover, such relaxations are accompanied by complete dephosphorylation of MLC. Phorbol 12, 13-dibutyrate, an exogenous activator of protein kinase C potentiates the endothelin induced contractions. Inactive phorbol ester, 4α-phorbol ester does not elicit any contractile response in the muscle. The down regulation of protein kinase C, on the other hand suppresses such potentiated contractile responses. These results suggest that endothelin-1 induced contractile tensions in tracheal smooth muscle are mediated by a mechanism that involves an activation of enzyme protein kinase C.     

Effects of NS1608 on MaxiK Channels in Smooth Muscle Cells from Urinary Bladder

Using the patch-clamp technique, we have characterized membrane currents in single detrusor smooth muscle cells from rat and human urinary bladder. From the voltage- and Ca²⁺-dependence of the current as well as the single channel conductance we conclude that rat and human urinary bladder smooth muscle cells express MaxiK channels. In smooth muscle cells from rat urinary bladder we tested the action of NS1608 on current through these MaxiK channels. Application of 10 μm NS1608 increased the amplitude of the current and this increase could be explained by a shift in the activation voltage of the MaxiK channels ∼100 mV towards more negative potentials. Charybdotoxin as well as paxilline, well known blockers of MaxiK channels, were able to reduce current through MaxiK channels in our cell preparation. In addition, application of 10 μm NS1608 hyperpolarized the membrane potential of the investigated cells. This hyperpolarization could be antagonized by the application of paxilline. We conclude that application of NS1608 results in the opening of MaxiK channels under physiological conditions that leads to a hyperpolarization of the cells. This hyperpolarization in turn could relax urinary bladder smooth muscle cells. MaxiK channels in these cells could therefore play a role in directly controlling muscle tone by regulating the membrane potential. This opens up the possibility of MaxiK channels being targets for the treatment of urge incontinence. Received: 19 July/Revised: 20 September 1999     

Beta2-integrins contribute to skeletal muscle hypertrophy in mice

We tested the contribution of β₂-integrins, which are important for normal function of neutrophils and macrophages, to skeletal muscle hypertrophy after mechanical loading. Using the synergist ablation model of hypertrophy and mice deficient in the common β-subunit of β₂-integrins (CD18^–/–), we found that overloaded muscles of wild-type mice had greater myofiber size, dry muscle mass, and total protein content compared with CD18^–/– mice. The hypertrophy in wild-type mice was preceded by elevations in neutrophils, macrophages, satellite cell/myoblast proliferation (5'-bromo-2'-deoxyuridine- and desmin-positive cells), markers of muscle differentiation (MyoD1 and myogenin gene expression and formation and size of regenerating myofibers), signaling for protein synthesis [phosphorylation of Akt and 70-kDa ribosomal protein S6 kinase (p70S6k)], and reduced signaling for protein degradation (decreased gene expression of muscle atrophy F box/atrogin-1). The deficiency in β₂-integrins, however, altered the accumulation profile of neutrophils and macrophages, disrupted the temporal profile of satellite cell/myoblast proliferation, reduced the markers of muscle differentiation, and impaired the p70S6k signaling, all of which could serve as mechanisms for the impaired hypertrophy in overloaded CD18^–/– mice. In conclusion, our findings indicate that β₂-integrins contribute to the hypertrophic response to muscle overload by temporally regulating satellite cells/myoblast proliferation and by enhancing muscle differentiation and p70S6k signaling. skeletal muscle growth; neutrophils; macrophages; compensatory hypertrophy     

P2Y1 receptors mediate inhibitory purinergic neuromuscular transmission in the human colon

Indirect evidence suggests that ATP is a neurotransmitter involved in inhibitory pathways in the neuromuscular junction in the gastrointestinal tract. The aim of this study was to characterize purinergic inhibitory neuromuscular transmission in the human colon. Tissue was obtained from colon resections for neoplasm. Muscle bath, microelectrode experiments, and immunohistochemical techniques were performed. 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate tetraammonium salt (MRS 2179) was used as a selective inhibitor of P2Y(1) receptors. We found that 1) ATP (1 mM) and adenosine 5'-beta-2-thiodiphosphate (ADPbetaS) (10 microM), a preferential P2Y agonist, inhibited spontaneous motility and caused smooth muscle hyperpolarization (about -12 mV); 2) MRS 2179 (10 microM) and apamin (1 microM) significantly reduced these effects; 3) both the fast component of the inhibitory junction potential (IJP) and the nonnitrergic relaxation induced by electrical field stimulation were dose dependently inhibited (IC(50) approximately 1 microM) by MRS 2179; 4) ADPbetaS reduced the IJP probably by a desensitization mechanism; 5) apamin (1 microM) reduced the fast component of the IJP (by 30-40%) and the inhibitory effect induced by electrical field stimulation; and 6) P2Y(1) receptors were localized in smooth muscle cells as well as in enteric neurons. These results show that ATP or a related purine is released by enteric inhibitory motoneurons, causing a fast hyperpolarization and smooth muscle relaxation. The high sensitivity of MRS 2179 has revealed, for the first time in the human gastrointestinal tract, that a P2Y(1) receptor present in smooth muscle probably mediates this mechanism through a pathway that partially involves apamin-sensitive calcium-activated potassium channels. P2Y(1) receptors can be an important pharmacological target to modulate smooth muscle excitability.     

Effect of exogenous ATP on canine jejunal smooth muscle

Intracellular recordings were made from the circular smooth muscle cells of the canine jejunum to study the effect of exogenous ATP and to compare the ATP response to the nonadrenergic, noncholinergic (NANC) inhibitory junction potential (IJP) evoked by electrical field stimulation (EFS). Under NANC conditions, exogenous ATP evoked a transient hyperpolarization (6.5 +/- 0.6 mV) and EFS evoked a NANC IJP (17 +/- 0.4 mV). Omega-conotoxin GVIA (100 nM) and a low-Ca(2+), high-Mg(2+) solution abolished the NANC IJP but had no effect on the ATP-evoked hyperpolarization. The ATP-evoked hyperpolarization and the NANC IJP were abolished by apamin (1 microM) and N(G)-nitro-L-arginine (100 microM). Oxyhemoglobin (5 microM) partially (38.8 +/- 5.5%) reduced the amplitude of the NANC IJP but had no effect on the ATP-evoked hyperpolarization. Neither the NANC IJP nor the ATP-evoked hyperpolarization was affected by P2 receptor antagonists or agonists, including suramin, reactive blue 2, 1-(N, O-bis-[5-isoquinolinesulfonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine , pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid, alpha, beta-methylene ATP, 2-methylthioadenosine 5'-triphosphate tetrasodium salt, and adenosine 5'-O-2-thiodiphosphate. The data suggest that ATP evoked an apamin-sensitive hyperpolarization in circular smooth muscle cells of the canine jejunum via local production of NO in a postsynaptic target cell.     

Activation by Angiotensin II of Ca2+-dependent K+ and Cl− Currents in Zona Fasciculata Cells of Bovine Adrenal Gland

The effects of angiotensin II (100 nm) on the electrical membrane properties of zona fasciculata cells isolated from calf adrenal gland were studied using the whole cell patch recording method. In current-clamp condition, angiotension II induced a biphasic membrane response which began by a transient hyperpolarization followed by a depolarization more positive than the control resting potential. These effects were abolished by Losartan (10⁻⁵ m), an antagonist of angiotensin receptors of type 1. The angiotensin II-induced transient hyperpolarization was characterized in voltage-clamp condition from a holding potential of −10 mV. Using either the perforated or the standard recording method, a transient outward current accompanied by an increase of the membrane conductance was observed in response to the hormonal stimulation. This outward current consisted of an initial fast peak followed by an oscillating or a slowly decaying plateau current. In Cl⁻-free solution, the outward current reversed at −78.5 mV, a value close to E _K. It was blocked by external TEA (20 mm) and by apamin (50 nm). In K⁺-free solution, the transient outward current, sensitive to Cl⁻ channel blocker DPC (400 μm), reversed at −52 mV, a more positive potential than E _Cl. Its magnitude changed in the same direction as the driving force for Cl⁻. The hormone-induced transient outward current was never observed when EGTA (5 mm) was added to the pipette solution. The plateau current was suppressed in nominally Ca²⁺-free solution (47% of cells) and was reversibly blocked by Cd²⁺ (300 μm) but not by nisoldipine (0.5–1 μm) which inhibited voltage-gated Ca²⁺ currents identified in this cell type. The present experiments show that the transient hyperpolarization induced by angiotensin II is due to Ca²⁺-dependent K⁺ and Cl⁻ currents. These two membrane currents are co-activated in response to an internal increase of [Ca²⁺] _i originating from intra- and extracellular stores. Received: 29 May 1997/Revised: 4 November 1997