Gastric Fundus Smooth Muscle Research Articles

Impaired gastric motility in the gastroesophageal reflux rat model: an in vitro study

Purpose The present study investigated the effects of acid and mixed reflux on the responsiveness of gastric smooth muscle in the gastroesophageal reflux (GER) rat model. Material and methods Three groups of rat were studied encompassing acid reflux, mixed reflux and sham operation. Acid reflux was induced by pyloric ligation (AR group) and mixed reflux was induced by jejunal ligation 1 cm distal to Treitz ligament (MR group). Similar surgical manipulations were carried out in the sham operated rats (SO group). Carbachol-, serotonin-, KCl-induced contractile response and nicotine-, sodium nitroprusside-, papaverine-induced relaxant response in isolated gastric fundus smooth muscle strips were determined using in vitro muscle technique 24 h after surgery. Results Isolated gastric fundus smooth muscle contractility to serotonin, carbachol or KCl was significantly reduced in the AR and MR groups with decreased Emax and pD 2 values compared with the SO group. Relaxant responses to nicotine was significantly increased in the AR and MR groups with increased Emax and pD 2 values compared with the SO group. Sodium nitroprusside and papaverine-induced-relaxant responses were similar in all of the groups and there was no change in agonist potency. Conclusion The present study indicates that decreased contractile and increased nicotine-induced relaxant response of the gastric smooth muscle in the surgically created GER model. These findings suggest that impaired gastric smooth muscle reactivity at least in part may play a role in gastric dysmotility in GER.

Gastric smooth muscle contractility changes in the esophageal atresia rat model: an in vitro study

Purpose: The aim of the study was to investigate the gastric smooth muscle reactivity in the Adriamycin-induced esophageal atresia (EA) rat model. Methods: The fetuses were divided into 3 groups. The control group was exposed to saline. The second group was comprised of fetuses that were exposed to Adriamycin but did not have EA (Adriamycin-no-EA group). The third group was comprised of fetuses that were exposed to Adriamycin and had EA (Adriamycin-EA group). Gastric fundus strips were studied in vitro for their contractile response to receptor activation in the 3 groups. Results: Contractile responses of gastric smooth muscle to carbachol and KCl were increased in the Adriamycin-EA group compared with the Adriamycin-no-EA group. Also serotonin-induced contractile response in the Adriamycin-EA group decreased compared with the Adriamycin-no-EA group. Relaxation of gastric smooth muscle strips to isoproterenol was comparably unaffected in the Adriamycin-EA and Adriamycin-no-EA groups. Likewise, no change in the response to agonist studies was observed between the control and Adriamycin-no-EA groups. The relaxant response to papaverine was not different in the 3 groups. Conclusions: This study found changes of receptor-dependent and receptor-independent contraction of the gastric fundus smooth muscle in the fetuses with EA. Therefore, impaired contractile responses may be, at least in part, a contributing factor in the abnormal gastric motility seen in EA.

Melatonin reduces non-adrenergic, non-cholinergic relaxant neurotransmission by inhibition of nitric oxide synthase activity in the gastrointestinal tract of rodents in vitro.

The aim of the present study was to investigate the effects of melatonin on non-adrenergic, non-cholinergic (NANC) relaxant neurotransmission in the gastrointestinal tract, which is mainly mediated by nitrergic and peptidergic mechanisms. Melatonin (10(-7)-10(-3) M) had no effect on the basal tonus of the rat gastric fundus smooth muscle. Relaxant responses following electrical stimulation(40 V; 0.5 ms pulse duration; 10 s stimulation duration) under NANC conditions on a 5-hydroxytryptamine (5-HT, 10(-7) M) contraction plateau were elicited at frequencies in the range of 0.5-16 Hz. Melatonin significantly reduced these inhibitory NANC responses (16 Hz without melatonin: -103 +/- 6.3%; melatonin 10(-5) M: -80.4 +/- 7.5%; melatonin 10(-4) M: -39.1 +/- 17.1%). Intracellular recording was carried out in a mouse colonic preparation. Electrical neural stimulation of the mouse colonic neurons caused biphasic intracellular hyperpolarization in smooth-muscle cells. The initial fast component is apamin-sensitive, and the following slow component is dependent on nitrergic mechanisms, as it is abolished in the presence of NG-nitro-L-arginine (L-NNA). Melatonin significantly reduced the nitric oxide-dependent slow component of neurally transmitted hyperpolarization, whereas the initial fast component was left unchanged. In a synaptosomal preparation of the enteric nervous system of rat intestine, enzymatic nitric oxide synthase (NOS) activity was significantly reduced by melatonin at concentrations ranging from 10(-7) to 10(-4) M (basal preparation including cofactors: 61.2 +/- 9.4 fmol/mg; melatonin 10(-4) M: 39.2 +/- 6.9 fmol/mg). Reverse transcriptase-polymerase chain reaction (RT-PCR) studies were conducted to investigate the melatonin receptors (mt(1), MT(2) and MT(3)) present in the esophagus, stomach and ileum of the rat. The presence of mt1 mRNA expression alone, but not of mRNA expression for MT(2) or MT(3), was demonstrated in the tissues. In conclusion, this study demonstrates that melatonin reduces the functional inhibitory NANC response. It shows that this effect may be the result of a reduction of the nitrergic component of the smooth-muscle inhibitory junction potential (IJP) and related to direct inhibition of NOS activity in enteric synaptosomes. The presence of mt1 receptor transcripts adds supportive evidence for a possible physiological role of melatonin within the enteric nervous system.

Endomorphins 1 and 2 reduce relaxant non-adrenergic, non-cholinergic neurotransmission in rat gastric fundus

It is now well established that opioids modulate cholinergic excitatory neurotransmission in the gastrointestinal tract. The aim of the present study was to characterize a possible effect of endomorphins on nonadrenergic, noncholinergic (NANC) relaxant neurotransmission in the rat gastric fundus in vitro. The drugs used in the experiments were the endogenous μ-opioid receptors (MORs) endomorphin 1 and 2 and the μ-opioid receptor antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2). CTAP left the basal tonus and the spontaneous activity of the preparation unchanged. Electrical field stimulation (EFS) under NANC conditions at frequencies ranging from 0.5 to 16 Hz caused a frequency-dependent relaxant response on the 5-hydoxytryptamine (5-HT) (10 −7 M) precontracted smooth-muscle strip. Both endomorphin 1 and endomorphin 2 significantly reduced this relaxation in a concentration-dependent manner. Endomorphin 1 proved to be more potent in reducing the relaxant responses. The endomorphin effects were significantly reversed by the MOR antagonist CTAP. CTAP itself did not influence the EFS-induced relaxation. In summary, these data provide evidence that the endogenous MOR agonists endomorphin 1 and 2 can reduce nonadrenergic, noncholinergic neurotransmission in the rat gastric fundus smooth muscle via a pathway involving MORs. The physiological relevance of these findings remains to be established, since the data presented suggest that the endomorphins act as neuromodulators within NANC relaxant neurotransmission.

Effects of some sterically hindered phenols on whole-cell Ca(2+) current of guinea-pig gastric fundus smooth muscle cells.

1. The aim of the present study was to investigate the effects of extracellular application of some sterically-hindered phenols, namely 3-t-butyl-4-hydroxyanisole (BHA), 3,5-di-t-butyl-4-hydroxyanisole (DTBHA) and the dimer of BHA, 2,2'-dihydroxy-3,3'-di-t-butyl-5,5'-dimethoxydiphenyl (DIBHA), on the whole-cell Ca(2+) current (I(Ca)) of freshly isolated smooth muscle cells from the guinea-pig gastric fundus, in the presence of a range of Ca(2+) concentrations (1 -- 5 mM) using the patch-clamp technique. The influx of Ca(2+) had characteristics of L-type I(Ca) (I(Ca(L))). 2. BHA as well as DTBHA inhibited I(Ca(L)) in a concentration-dependent manner, during depolarization to 10 mV from a holding potential of -50 mV. Bath application of BHA (50 microM) and DTBHA (30 microM) decreased I(Ca(L)) by 48.9% and 45.2%, respectively. This inhibition was only partially reversible. In contrast, DIBHA (up to 50 microM) was devoided of effects on I(Ca(L)). 3. BHA inhibition of I(Ca(L)) was voltage-dependent and inversely related to the external concentration of Ca(2+). On the other hand, DTBHA inhibition was only voltage-dependent. 4. BHA and DTBHA shifted the voltage range of the steady-state inactivation curve to more negative potentials by 8 mV at the mid-potential of the curve, without affecting the activation curve. Furthermore, BHA and DTBHA did not modify the time-course of the current decay. 5. We conclude that the inhibition of I(Ca(L)) by BHA and DTBHA is qualitatively similar to that of a Ca(2+) channel blocker and is characterized by the stabilizing effect of the inactivated state of the channel.

Inhibition of small conductance K+-channels attenuated melatonin-induced relaxation of serotonin-contracted rat gastric fundus

The aim of this study was to investigate the effects of melatonin on rat gastric fundus smooth muscle. Melatonin (10-4 to10-3 M) had no effect on the basal tone of gastric smooth muscle. After precontraction with carbachol (10-6 M) or serotonin (10-7 M), melatonin caused a concentration dependent inhibitory action. The half maximal effect on serotonin-induced contraction was found with 1.12 beta 0.86 beta 10-5 M of melatonin. Increasing concentrations of melatonin (10-5 to 10-3 M) resulted in a right shift of the serotonin concentration response curve (10-10 to10-5 M). This inhibitory effect of melatonin was partially blocked in the presence of apamin (10-10 to 10-7 M), a specific blocker of the small conductance calcium-dependent potassium channel, but not in the presence of other potassium channel blockers like charybdotoxin (10-8 M), glibenclamide (10-5 M), or tetraethylammonium (ODQ, 10-4 M). The inhibitory effect was not changed in the presence of the neuronal blocker tetrodotoxin (10-6 M), the selective P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (3 × 10-5 M), the nitric-oxide synthase inhibitor N-nitro-L-arginine (3 × 10-4 M), or the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (10-4 M), suggesting that neither the purinergic, nitrergic, nor guanylate cyclase pathways were involved. We further investigated inhibitory responses to electrical field stimulation (EFS) at different frequencies under non-adrenergic, non-cholinergic (NANC) conditions on a serotonin-induced contraction in the presence of melatonin (10-5 to 10-4 M). Melatonin significantly reduced these inhibitory NANC responses in higher (8-32 Hz), but not lower (05-4 Hz), frequencies (16 Hz without melatonin, 103 ± 6.3%; melatonin 10-5 M, 80.4 ± 7.5%; melatonin 10-4 M, 39.1 ± 17.1%). Melatonin had no effect on contractile responses induced by EFS under basal tone. These results demonstrate that the inhibitory effect of melatonin in rat gastric fundus smooth muscle is apamin sensitive, but is not affected by other potassium channel blockers. This suggests that melatonin may be another transmitter candidate for the apamin sensitive responses within the gastrointestinal tract.Key words: melatonin, smooth muscle, apamin, NANC-inhibition, gastric, potassium channels.

Evidence for VIP(1)/PACAP receptors in the afferent pathway mediating surgery-induced fundic relaxation in the rat.

We previously reported activation of an inhibitory adrenergic and a non-adrenergic non-cholinergic (NANC) pathway during abdominal surgery relaxing the rat gastric fundus. In the present study, we investigated the possible role of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) in the NANC part of the surgery-induced fundic relaxation. The effect of the NO biosynthesis inhibitor N(G)-nitro-L-arginine (L-NOARG), the non-selective VIP receptor antagonist [D-p-Cl-Phe(6),Leu(17)]-VIP and the selective VIP(1) receptor antagonist [Acetyl-His(1),D-Phe(2),Lys(15),Arg(16), Leu(17)]-VIP was investigated on the non-adrenergic fundic relaxation induced by manipulation of the small intestine followed by resection of the caecum. Guanethidine partly reduced the manipulation-induced fundic relaxation. Addition of L-NOARG reduced this non-adrenergic component, whereas the non-selective VIP receptor antagonist had no significant effect. Combination of L-NOARG and the non-selective VIP antagonist however further reduced the relaxation to manipulation. The selective VIP(1) receptor antagonist reduced the mean and maximal relaxation induced by abdominal surgery in the presence of guanethidine. When combined with L-NOARG, the relaxation of the gastric fundus was almost completely abolished. The VIP(1) receptor antagonist alone had no significant effect on the mean and maximal relaxation, but enhanced recovery of fundic tone. In conclusion, as VIP(1) receptors are not present in the rat gastric fundus, these results suggest that the NANC inhibitory pathway activated during abdominal surgery involves VIP(1) receptors, most likely in the afferent limb. The inhibitory neurotransmitters released at the level of the gastric fundus smooth muscle are NO and a substance different from VIP.

Interplay between nitric oxide and vasoactive intestinal polypeptide in the pig gastric fundus smooth muscle

The aim of this study was to investigate the exact mechanism of interaction between nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) as inhibitory non-adrenergic non-cholinergic (NANC) neurotransmitters in isolated smooth muscle cells and smooth muscle strips of the pig gastric fundus. In isolated smooth muscle cells, the maximal relaxant effect of VIP (10 −9 M) was inhibited by 94% by the NO synthase (NOS) inhibitor N G-nitro- l-arginine ( l-NA, 10 −4 M) and by 85% by the inducible NOS (iNOS)-selective inhibitor N-(3-(aminomethyl)-benzyl)acetamide (1400W; 10 −6 M). The relaxant effect of VIP was reduced by more than 70% by the guanylyl cyclase inhibitor 1 H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ; 10 −6 M), the glucocorticoid dexamethasone (10 −5 M) and three protein kinase A inhibitors: ( R)- p-cyclic adenosine-3′,5′-monophosphothioate (( R)- p-cAMPS; 10 −6 M), {(8 R,9 S,11 S)-(−)-9-hydroxy-9- n-hexylester-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1 H,8 H,11 H-2,7 b,11 a-triazadibenzo[ a, g]cycloocta[ cde]-trin-den-1-one} (KT5720; 10 −6 M) and N-(2-( p-bromo-cinnamylamino)ethyl))-5-isoquinoline sulfonamide dihydrochloride (H-89; 10 −5 M). In contrast, no influence of the NOS inhibitors, ODQ, dexamethasone, nor the protein kinase A inhibitors could be observed on the relaxant effect of VIP in smooth muscle strips. These data demonstrate that the experimental method completely changes the influence of NOS inhibitors on the relaxant effect of VIP in the pig gastric fundus. The isolation procedure of the smooth muscle cells might induce iNOS that can be activated by VIP.

Impaired relaxation of stomach smooth muscle in mice lacking cyclic GMP-dependent protein kinase I.

1. Guanosine 3', 5'-cyclic monophosphate (cyclic GMP)-dependent kinase I (cGKI) is a major receptor for cyclic GMP in a variety of cells. Mice lacking cGKI exhibit multiple phenotypes, including severe defects in smooth muscle function. We have investigated the NO/cGMP- and vasoactive intestinal polypeptide (VIP)/adenosine 3', 5'-cyclic monophosphate (cyclic AMP)-signalling pathways in the gastric fundus of wild type and cGKI-deficient mice. 2. Using immunohistochemistry, similar staining patterns for NO-synthase, cyclic GMP- and VIP-immunoreactivities were found in wild type and cGKI-deficient mice. 3. In isolated, endothelin-1 (3 nM - 3 microM)-contracted, muscle strips from wild type mice, electrical field stimulation (1 - 16 Hz) caused a biphasic relaxation, one initial rapid, followed by a more slowly developing phase. In preparations from cGKI-deficient mice only the slowly developing relaxation was observed. 4. The responses to the NO donor, SIN-1 (10 nM - 100 microM), and to 8-Br-cyclic GMP (10 nM - 100 microM) were markedly impaired in strips from cGKI-deficient mice, whereas the responses to VIP (0.1 nM - 1 microM) and forskolin (0.1 nM - 1 microM) were similar to those in wild type mice. 5. These results suggest that cGKI plays a central role in the NO/cGMP signalling cascade producing relaxation of mouse gastric fundus smooth muscle. Relaxant agents acting via the cyclic AMP-pathway can exert their effects independently of cGKI.

Inhibition of small conductance K<sup>+</sup>-channels attenuated melatonin-induced relaxation of serotonin-contracted rat gastric fundus

The aim of this study was to investigate the effects of melatonin on rat gastric fundus smooth muscle. Melatonin (10(-4) to 10(-3) M) had no effect on the basal tone of gastric smooth muscle. After precontraction with carbachol (10(-6) M) or serotonin (10(-7) M), melatonin caused a concentration dependent inhibitory action. The half maximal effect on serotonin-induced contraction was found with 1.12 +/- 0.86 x 10(-5) M of melatonin. Increasing concentrations of melatonin (10(-5) to 10(-3) M) resulted in a right shift of the serotonin concentration response curve (10(-10) to 10(-5) M). This inhibitory effect of melatonin was partially blocked in the presence of apamin (10(-10) to 10(-7) M), a specific blocker of the small conductance calcium-dependent potassium channel, but not in the presence of other potassium channel blockers like charybdotoxin (10(-8) M), glibenclamide (l0(-5) M), or tetraethylammonium (ODQ, 10(-4) M). The inhibitory effect was not changed in the presence of the neuronal blocker tetrodotoxin (10(-6) M), the selective P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (3 x 10(-5) M), the nitric-oxide synthase inhibitor N-nitro-L-arginine (3 x 10(-4) M), or the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (10(-4) M), suggesting that neither the purinergic, nitrergic, nor guanylate cyclase pathways were involved. We further investigated inhibitory responses to electrical field stimulation (EFS) at different frequencies under non-adrenergic, non-cholinergic (NANC) conditions on a serotonin-induced contraction in the presence of melatonin (10)-5 to 10(-4) M). Melatonin significantly reduced these inhibitory NANC responses in higher (8-32 Hz), but not lower (05-4 Hz), frequencies (16 Hz without melatonin, 103 +/- 6.3%; melatonin 10(-5) M, 80.4 +/- 7.5%; melatonin 10(-4) M, 39.1 +/- 17.1%). Melatonin had no effect on contractile responses induced by EFS under basal tone. These results demonstrate that the inhibitory effect of melatonin in rat gastric fundus smooth muscle is apamin sensitive, but is not affected by other potassium channel blockers. This suggests that melatonin may be another transmitter candidate for the apamin sensitive responses within the gastrointestinal tract.

Myorelaxant activity of 2- t-butyl-4-methoxyphenol (BHA) in guinea pig gastric fundus

This study investigates the mechanism whereby the antioxidant 2- t-butyl-4-methoxyphenol (BHA) relaxes guinea pig gastric fundus smooth muscle. In circular smooth muscle strips, 10 μM cyclopiazonic acid, a specific inhibitor of sarcoplasmic reticulum Ca 2+-ATPase, induced a prolonged rise in tension which depended on the presence of extracellular Ca 2+. BHA (pIC 50=5.83), sodium nitroprusside (6.85), isoproterenol (7.69) and nifedipine (8.02), but not 2,6-di- t-butyl-4-methoxyphenol (DTBHA) (up to 30 μM), relaxed muscle strips contracted with cyclopiazonic acid. Methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate (Bay K 8644) (1 μM) antagonised the nifedipine- but not the BHA-induced relaxation. Nifedipine and isoproterenol (10 μM) caused a decrease in spontaneous tone, but did not counteract the subsequent rise in tension elicited by 10 μM cyclopiazonic acid. Conversely, 100 μM BHA and 100 μM sodium nitroprusside not only significantly reduced spontaneous tone but also markedly impaired the response of the muscles to cyclopiazonic acid. DTBHA failed to show either effect. When added to preparations completely relaxed by 100 μM BHA, 10 mM tetraethylammonium still elicited nifedipine-sensitive tonic and phasic contractions in the presence or absence of 10 μM cyclopiazonic acid. BHA and DTBHA inhibited, in a concentration-dependent manner, the Ca 2+-promoted contraction of strips depolarised by 10 mM tetraethylammonium. The BHA antagonism showed a non-competitive profile while that of DTBHA was competitive. In muscle strips at rest, 10 μM BHA caused a significant increase in tissue cAMP concentration, leaving cGMP unmodified. To conclude, the myorelaxant action of BHA on gastric fundus smooth muscle appears to be mediated partly by an increase in cAMP levels and partly by inhibition of Ca 2+ influx from the extracellular space.

Comparison of Relaxations Evoked by Photoactivation of NO-Containing Compounds and Nitrergic Nerve Stimulation in 5-Hydroxytryptamine- and Potassium-Contracted Rat Gastric Fundus

1. The aim of the present study was to further investigate our earlier proposal of liberation of nitric oxide (NO) by photoactivation of molecules containing NO or NO 2, which in turn relaxes gastric smooth muscle, and to determine whether presynaptic- and/or postsynaptic NO-mediated relaxation is affected differently by the degree of membrane depolarization in rat gastric fundus smooth muscle. 2. During contraction of rat gastric fundus with 5-hydroxytryptamine (5-HT, 10 μM), low (K +, 25 mM) and high potassium (K +, 65.4 mM), relaxation responses to nitrergic nerve stimulation, photoactivation of caged NO compounds (streptozotocin [STZ], N ω-nitro- L-arginine-methylacetate [ L-NAME], N ω-nitro- D-arginine-methylacetate [ D-NAME]), and sodium nitroprusside (SNP) were compared. 3. Nitrergic nerve (presynaptic) stimulation and photoactivation (postsynaptic) of all caged NO compounds produced rapid, transient and reversible relaxation of 5-HT and low-K +-contracted tissues. However, when contractions were induced by high K +, the relaxation induced by nerve stimulation was abolished, whereas relaxations induced by photoactivated NO compounds were significantly ( P<0.01) reduced. 4. The relaxation induced by sodium nitroprusside (SNP), but not papaverine, was also diminished in high-K +-contracted tissues. The magnitude of photoactivated NO-induced relaxation was related to the amount of NO release, light intensity and concentration of compounds. 5. The evidence that photoactivated NO-induced relaxation is mediated by cGMP comes from the observation that zaprinast, but not forskolin, potentiated the relaxation. 6. It is concluded that rat gastric smooth muscle relaxes to photoactivation of NO or NO 2-carrying molecules via NO, and it appears that degree of membrane depolarization may be a critical factor in dissociating the response to presynaptic- and postsynaptic NO-mediated relaxation in this muscle.

The role of sarcoplasmic reticulum and sarcoplasmic reticulum Ca2+-ATPase in the smooth muscle tone of the cat gastric fundus.

Circular smooth muscle strips isolated from cat gastric fundus were studied in order to understand whether the sarcoplasmic reticulum (SR) and SR Ca2+-ATPase could play a role in the regulation of the muscle tone. Cyclopiazonic acid (CPA), a specific inhibitor of SR Ca2+-ATPase, caused a significant and sustained increase in muscle tone, depending on the presence of extracellular Ca2+. Nifedipine and cinnarizin only partially suppressed the CPA-induced tonic contraction. Bay K 8644 antagonized the relaxant effect of nifedipine in CPA-contracted fundus. Nitric-oxide-releasing agents sodium nitroprusside and 3-morpholino-sydnonimine completely suppressed the CPA-induced tonic contraction. The blockers of Ca2+-activated K+ channels, tetraethylammonium, charybdotoxin and/or apamin, decreased the contractile effect of CPA. Vanadate increased the tone but did not change significantly the effect of CPA. CPA exerted its contractile effect even when Ca2+ influx was triggered through the Na+/Ca2+ exchanger and the other Ca2+ entry pathways were blocked. Thapsigargin, another specific SR Ca2+-ATPase inhibitor, also increased the muscle tone. The effect of thapsigargin was completely suppressed by sodium nitroprusside and 3-morpholino-sydnonimine and partially by nifedipine. In conclusion, under conditions when the SR Ca2+-ATPase is inhibited, the tissue develops a strong tonic contraction and a large part of this is mediated by Ca2+ influx presumably via nifedipine-sensitive Ca2+ channels. This study suggests the important role of SR Ca2+-ATPase in the modulation of the muscle tone and the function of SR as a "buffer barrier" to Ca2+ entry in the cat gastric fundus smooth muscle.

Alteration of contractile properties to serotonin in gastric fundus smooth muscle isolated from streptozotocin (STZ)-induced diabetic rats.

Contractile responses to serotonin (5-HT) of fundic smooth muscle strips isolated from both control and streptozotocin (STZ)-induced diabetic rats were investigated. Contrary to carbachol (CCh) which causes contractile hyperactivity in DM, 5-HT response tended to decrease in DM compared to that of the control. Pindolol (10(-5)M) increased the value of EC50 of the concentration-response to 5-HT about 2.5 times in both the control and DM. After treatment with pindolol, the maximal tension to 5-HT in DM significantly decreased compared to that of the control. Pindolol showed no effect on the contractile response to CCh. Pindolol significantly inhibited the relaxation caused by isoproterenol in DM more than in the control. Mianserin (10(-5) M) increased the EC50 of the response to 5-HT about 2-2.5 times in both groups, but did not cause a significant difference between the control and DM. The Ca(2+)-induced contraction caused hyperreactivity in DM in the presence of 10(-6) M CCh, but that in DM was not significantly different from the control in the presence of 10(-6) M 5-HT. Pretreatment of phorbol 12-myristate 13-acetate (PMA, 10(-5) M) significantly attenuated the response to 5-HT in the control, but not in DM. Results suggest that the contractile response to 5-HT in DM is related to the altered Ca2+ signal transduction system via disturbed protein kinase C (PKC) activity, and that there are alterations of receptor characteristics and of the density in 5-HT receptor subtypes, especially 5-HT1A, during DM development.

P-593 - Nitric oxide synthase induction and relaxation in lipopolysaccharide-treated gastric fundus muscle of rats

Abstract To investigate whether L-arginine/nitric oxide (NO) pathway activated after treatment with lipopolysaccharide (LPS) could relax the gastric fundus smooth muscle, we made functional examinations and measured NO synthase activity by the conversion of radiolabelled L-arginine to L-citrulline in rat gastric fundus strips treated with LPS in vitro. L-arginine caused a relaxation of the mucosa-free gastric fundus strips which had been treated with LPS for 6 h in vitro and then contracted by PGF2 α beforehand. This relaxation was partially reversed by N G -nitro-L-arginine (a nitric oxide synthase inhibitor) or methylene blue (a soluble guanylate cyclase inhibitor). Ca 2+ -independent NO synthase activity was induced after LPS-treatment. Co-incubation with LPS and cycloheximide for 6 h inhibited the relaxation to L-arginine and the induction of NO synthase. On the other hand, Ca 2+ -dependent NO synthase activity was decreased after LPS-treatment. These results strongly suggest that Ca 2+ -independent NO synthase is induced by endotoxin in the gastric fundus muscle, resulting in inhibition of the contractile response.

Nitric oxide synthase induction and relaxation in lipopolysaccharide-treated gastric fundus muscle of rats

To investigate whether L-arginine/nitric oxide (NO) pathway activated after treatment with lipopolysaccharide (LPS) could relax the gastric fundus smooth muscle, we made functional examinations and measured NO synthase activity by the conversion of radiolabelled L-arginine to L-citrulline in rat gastric fundus strips treated with LPS in vitro. L-arginine caused a relaxation of the mucosa-free gastric fundus strips which had been treated with LPS for 6 h in vitro and then contracted by PGF2 α beforehand. This relaxation was partially reversed by N G-nitro-L-arginine (a nitric oxide synthase inhibitor) or methylene blue (a soluble guanylate cyclase inhibitor). Ca 2+-independent NO synthase activity was induced after LPS-treatment. Co-incubation with LPS and cycloheximide for 6 h inhibited the relaxation to L-arginine and the induction of NO synthase. On the other hand, Ca 2+-dependent NO synthase activity was decreased after LPS-treatment. These results strongly suggest that Ca 2+-independent NO synthase is induced by endotoxin in the gastric fundus muscle, resulting in inhibition of the contractile response.

Developmental Changes in Gastric Fundus Smooth Muscle Contractility and Involvement of Extracellular Calcium in Fetal and Adult Guinea Pigs

Delayed gastric emptying is a common problem in preterm infants. The factors underlying this gastroparesis remain unsettled but may involve immaturity of smooth muscle contraction. The present study was designed to test this hypothesis. Muscle strips from the gastric fundus of fetal and adult guinea pigs were studied in vitro for their contractile response to receptor activation (acetylcholine and bethanechol) and membrane depolarization (potassium chloride). The dose-response curves were analyzed for differences in active force development (kg/cm2). The role of extracellular calcium (Ca2+) in the contractile responses was determined by contracting the tissues in a zero-Ca2+ physiologic saline solution and in the presence of nifedipine, a voltage-dependent Ca2+ channel blocker. The results demonstrate the following: 1) tissues from adult animals developed significantly more active force when tested with acetylcholine, bethanechol, and potassium chloride; 2) tissues from the fetal animals were relatively unresponsive to contraction with potassium chloride compared with the adult; and 3) both nifedipine and incubation in a zero-Ca2+ physiologic saline solution had a significantly greater inhibitory effect on the contractions of adult than fetal muscle strips. Our data indicate that smooth muscle in the gastric fundus develops increasing force with maturation. The increased contractility in the adult fundus appears to be due to an increased involvement of extracellular calcium influx, in part through voltage-dependent Ca2+ channels.

Contractile hyperreactivity and alteration of PKC activity in gastric fundus smooth muscle of diabetic rats

Contraction dose dependently induced in gastric smooth muscle of diabetic rats by Bay K 8644 in the presence of 20 mM KCl was about two times that induced in controls, and was inhibited more than 50% by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). Contraction was caused in diabetics but usually not in controls by 10 −5 M phorbol 12-myristate 13-acetate (PMA). In diabetics, this contraction was about 2.5 times that in controls. Protein kinase C (PKC) activity in the soluble fraction was depressed by H-7 or staurosporine, and depended on PMA concentration, but was greater in diabetics than in controls at any PMA concentration. PKC activity in the soluble fraction was inhibited by lower Ca 2+ concentration, and was greater in diabetics than in controls. Affinity and density of binding sites of a Ca 2+ channel antagonist ligand, [ 3H]PN200-110, were the same in plasma membrane-enriched fractions isolated from either controls or diabetic preparations. Thus, hyperreactivity in diabetic fundus may depend, in part, on alteration of PKC properties, but not on the density of Ca 2+ channels.

P-168 - Changes of protein kinase C activity in gastric fundus smooth muscle in experimental diabetic rats

P-168 - Changes of protein kinase C activity in gastric fundus smooth muscle in experimental diabetic rats

Relations between contractile responses and β-adrenoceptors in gastric fundus of diabetic rats

Contractile responses to norepinephrine (NE), and the population of β-adrenoceptors, were determined in gastric fundus smooth muscle from rats with diabetes induced by streptozotocin(STZ), and age-matched controls. Relaxation and/or contraction of fundus strips of controls and diabetics were induced by 10 −5M NE. Responses to NE were mainly relaxation in gastric fundus isolated from controls, and contraction in fundus isolated from diabetics. Contraction was blocked by 10 −8M prazosin and relaxation was blocked by 10 −6 M propranolol. Relaxation by isoproterenol of contraction induced by 10 −6M acetylcholine was significantly less in fundus from diabetics than in that from controls. The number of β-adrenoceptors, measured with [ 125I]iodocyanopindorol as a ligand, was significantly less in gastic fundus membrane isolated from diabetics than in that from controls, but affinity was no different. The level of plasma catecholamine was higher in diabetics than in controls. Results suggest that depression of gastric fundus relaxation and increase of contraction by NE in diabetics could be due to fewer β-adrenoceptor binding sites caused by down-regulation by higher catecholamine level in diabetic rats.