Endogenous interstitial adenosine in isolated myenteric neural networks varies inversely with prevailing PO2.
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Isolated myenteric ganglion networks were used in a perifusion protocol to characterize the response of interstitial adenosine levels to changes in prevailing PO2. The biological activity of such adenosine was assessed using inhibition of release of substance P (SP) as a functional measure of adenosine activity, and the effect of altered O2 tension on both spontaneous and elevated extracellular K+ concentration-evoked SP release from networks was determined over a range of PO2 values from hypoxic (PO2 = 54 mmHg) to hyperoxic (PO2 = 566 mmHg). Release of SP was found to be sensitive to PO2, and a linear graded relationship was obtained. Perifusion in the additional presence of the adenosine A1-receptor-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) revealed considerable adenosinergic inhibition with an inverse exponential relationship and hyperoxic threshold PO2. Disinhibition of evoked SP release by DPCPX in the absence of TTX was double that observed in its presence, indicating a neural source for some of the adenosine released during hypoxia. A postulated neuroprotective role for adenosine is consistent with the demonstrated relationship between interstitial adenosine and prevailing O2 tension. (+info)
Downregulation of Galphaq-11 protein expression in guinea pig antral and colonic circular muscle during pregnancy.
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Pregnancy has an inhibitory effect on motility of the gastrointestinal tract. The present study was designed to examine the mechanisms responsible for antral and colonic hypomotility in pregnant guinea pigs. Circular smooth muscle cells from the antrum and left colon were isolated by enzymatic digestion with collagenase from pregnant and nonpregnant guinea pigs. Contractile responses to agonists were expressed as percent shortening from resting cell length. The function of G proteins in antral and colonic circular smooth muscle was assessed by [35S]guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding induced by CCK-8 and G protein quantitation. The contraction of antral and colonic circular smooth muscle from pregnant guinea pigs was reduced in response to CCK-8 and to GTPgammaS but was normal in response to KCl and D-myo-inositol 1,4,5-trisphosphate compared with nonpregnant animals. The stimulation of [35S]GTPgammaS binding to Galphaq-11 induced by 1 microM CCK-8 was significantly lower in antral and colonic circular smooth muscle from pregnant guinea pigs than that in controls. Furthermore, Western blot analysis showed a decreased Galphaq-11 and an increased Gsalpha protein content in both tissues during pregnancy. It is concluded that pregnancy appears to impair gastrointestinal circular smooth muscle contractility by downregulating G proteins such as Galphaq-11 protein, which mediates muscle contraction, and upregulating Gsalpha protein, which mediates muscle relaxation. (+info)
Vagus nerve modulates secretin binding sites in the rat forestomach.
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Secretin is well known for its inhibitory action on gastric motility. It has been reported that secretin in a physiological dose inhibits gastric motility through mediation by the vagal afferent pathway. Secretin also elicited relaxation of carbachol-stimulated rat forestomach muscle strips by binding to its receptors, suggesting a direct action on this peripheral tissue. We hypothesized that vagal input may affect the action of secretin by modulating the level of secretin receptor in the forestomach. Several treatments, including vagal ligation, vagotomy, perivagal application of capsaicin or colchicine, intravenous infusion of tetrodotoxin, and intraperitoneal injection of atropine, were performed to investigate their effects on secretin receptor binding to forestomach membranes. Specific binding of 125I-labeled secretin to forestomach membranes was significantly decreased (45%) by vagal ligation, vagotomy (50%), or perivagal colchicine treatment (40%). On the contrary, specific binding of 125I-secretin was not affected by perivagal capsaicin treatment, intravenous infusion of tetrodotoxin, or intraperitoneal injection of atropine. By Scatchard analysis of the binding data, the capacity of the high-affinity binding sites in forestomach membranes was found to decrease significantly after vagal ligation compared with membranes from the sham-operated group. However, the affinity at the high-affinity binding sites, the binding parameters of the low-affinity binding sites, and binding specificity were not changed. Vagal ligation but not perivagal capsaicin treatment reduced the inhibitory effect of secretin on bethanechol-stimulated contraction of isolated forestomach muscle strips, causing a right shift in the dose-response curve. These results suggest that vagal input through axonal transport plays a significant role on secretin action by modulating the capacity of secretin binding sites (but not affinity or specificity), at least in rat forestomach. (+info)
Gialpha but not gqalpha is linked to activation of p21(ras) in human airway smooth muscle cells.
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Airway smooth muscle hypertrophy contributes to the narrowing of asthmatic airways. Activation of the mitogen-activated protein kinases is an important event in mediating cell proliferation. Because the monomeric G protein p21(ras) is an important intermediate leading to activation of mitogen-activated protein kinases, we questioned which heterotrimeric G protein-coupled receptors were linked to the activation of p21(ras) in cultured human airway smooth muscle and which of the heterotrimeric G protein subunits (alpha or betagamma) transmitted the activation signal. Carbachol and endothelin-1 increased GTP-bound p21(ras) in a pertussis toxin-sensitive manner [ratio of [32P]GTP to ([32P]GTP + [32P]GDP): control, 30 +/- 1.7; 3 min of 1 microM carbachol, 39 +/- 1.1; 3 min of 1 microM endothelin-1, 40 +/- 1.2], whereas histamine, bradykinin, and KCl were without effect. Transfection of an inhibitor of the G protein betagamma-subunit [the carboxy terminus (Gly495-Leu689) of the beta-adrenoceptor kinase 1] failed to inhibit the carbachol-induced activation of p21(ras). These data suggest that Gi- but not Gq-coupled receptors activate p21(ras) in human airway smooth muscle cells, and this effect most likely involves the alpha-subunit. (+info)
Two heads of myosin are better than one for generating force and motion.
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Several classes of the myosin superfamily are distinguished by their "double-headed" structure, where each head is a molecular motor capable of hydrolyzing ATP and interacting with actin to generate force and motion. The functional significance of this dimeric structure, however, has eluded investigators since its discovery in the late 1960s. Using an optical-trap transducer, we have measured the unitary displacement and force produced by double-headed and single-headed smooth- and skeletal-muscle myosins. Single-headed myosin produces approximately half the displacement and force (approximately 6 nm; 0.7 pN) of double-headed myosin (approximately 10 nm; 1.4 pN) during a unitary interaction with actin. These data suggest that muscle myosins require both heads to generate maximal force and motion. (+info)
The Rho-related protein Rnd1 inhibits Ca2+ sensitization of rat smooth muscle.
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1. The small GTP-binding Rho proteins are involved in the agonist-induced Ca2+ sensitization of smooth muscle. The action and the expression of Rnd1, a new member of the Rho protein family constitutively bound to GTP, has been studied in rat smooth muscle. 2. Recombinant prenylated Rnd1 (0.01-0.1 mg ml-1) dose dependently inhibited carbachol- and GTPgammaS-induced Ca2+ sensitization in beta-escin-permeabilized ileal smooth muscle strips but had no effect on the tension at submaximal [Ca2+] (pCa 6.3). Rnd1 inhibited GTPgammaS-induced tension without shifting the dose-response curves to GTPgammaS. 3. pCa-tension relationships were not modified by Rnd1 and the rise in tension induced through the inhibition of myosin light chain phosphatase by calyculin A was not affected by Rnd1. 4. The Ca2+ sensitization induced by recombinant RhoA was completely abolished when RhoA and Rnd1 were applied together. 5. Rnd1 was expressed at a low level in membrane fractions prepared from intestinal or arterial smooth muscles. The expression of Rnd1 was strongly increased in ileal and aortic smooth muscle from rats treated with progesterone or oestrogen. Progesterone-treated ileal muscle strips showed a decrease in agonist-induced Ca2+ sensitization. 6. The present study shows that (i) Rnd1 inhibits agonist- and GTPgammaS-induced Ca2+ sensitization of smooth muscle by specifically interfering with a RhoA-dependent mechanism and (ii) an increase in Rnd1 expression may account, at least in part, for the steroid-induced decrease in agonist-induced Ca2+ sensitization. (+info)
Role of PI3-kinase in the development of interstitial cells and pacemaking in murine gastrointestinal smooth muscle.
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1. Development of the pacemaker system in the small intestine depends upon signalling via tyrosine kinase (Kit) receptors. The downstream pathways initiated by Kit in interstitial cells of Cajal (ICC) have not been investigated. Wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002), inhibitors of phosphatidylinositol 3'-kinase (PI3-kinase), were used to test the involvement of this pathway in the development and maintenance of ICC and electrical rhythmicity in the murine small intestine. 2. ICC and electrical slow waves were present in the murine jejunum at birth. ICC and electrical rhythmicity continued to develop in neonates such that adult activity was recorded after 1 week. Development of ICC and rhythmicity were maintained in organ culture. 3. Wortmannin or LY 294002 inhibited the development of slow waves and blocked rhythmicity within 2-4 days. Loss of slow waves was preceded by disappearance of Kit-positive cells from the myenteric (IC-MY) and deep muscular plexus (IC-DMP) regions. Wortmannin or LY 294002 had no acute effect on slow waves. 4. Muscles from older animals (day 10-day 30) developed resistance to wortmannin treatment, but when the exposure to wortmannin was increased to 35 days, damage to ICC networks and electrical dysrhythmias were observed. 5. PI3-kinase appears to be a critical downstream signalling element linking Kit receptors to ICC development and maintenance of phenotype. ICC are more sensitive to Kit or PI3-kinase blockade at birth, but the importance of the PI3-kinase signalling in the maintenance of ICC persists into adulthood. Interference with PI3-kinase signalling in immature or adult animals could result in disruption of ICC and gastrointestinal dysrhythmias. (+info)
Subchronic exposure to diisocyanates increases guinea pig tracheal smooth muscle responses to acetylcholine.
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OBJECTIVE: In order to study the threshold concentrations of isocyanates (IC) for induction of lung disorders, constrictive responses of tracheal smooth muscles to acetylcholine (ACH) in guinea pigs with and without diisocyanate [toluene diisocyanate (TDI), hexamethylene diisocyanate (HDI) and diphenylmethane diisocyanate (MDI)] exposure were investigated. METHODS: An IC-induced increase in smooth muscle responsiveness was studied by measuring cumulative ACH dose responses (10(-10) to 10(-4) M ACH). Basal ACH dose-response curves, measured twice in intervals of 1 h using tracheal preparations of 11 guinea pigs previously not exposed to IC, were reproducible. RESULTS: Subchronic in vivo exposures to TDI, HDI, and MDI atmospheres of 10 and 20 parts per billion (ppb) on 5 consecutive days led to significantly (p < 0.05) increased ACH responsiveness of tracheal smooth muscle, whereas concentrations of 2.5 and 5 ppb were not effective. Exposure to HDI atmospheres of 10 ppb for 1, 2, 4, or 8 weeks resulted in a time-dependent increase in ACH responses (p < 0.05) of guinea pig tracheal smooth muscle. Increased tracheal muscle responses to ACH were transient since tracheal preparations from animals exposed to 10 and 20 ppb MDI for 4 weeks and with an exposure-free interval of 8 weeks before preparation did not show enlarged ACH responses, which were present in preparations at the end of the exposure period (p < 0.05). Exposure to low IC concentrations as present in workplaces cause increased ACH responsiveness of guinea pig tracheal smooth muscle. The increased responsiveness of the airways seems to be largely reversible, since normal responses were found after 8 weeks of IC avoidance. CONCLUSION: Reversibility of IC-induced airway hyperresponsiveness is of great occupational and preventive medical importance. Workers with acquired airway hyperresponsiveness might escape lung damage if the changes are detected in an early stage before alterations in lung function are in a chronic stage. (+info)