Guinea Pig Gastric Smooth Muscle Research Articles

Effects of Polyamines on Contractility of Guinea-Pig Gastric Smooth Muscle

This study was designed to investigate the effects of polyamines on mechanical contraction and voltage-dependent calcium current (VDCC) of guinea-pig gastric smooth muscle. Mechanical contraction and calcium channel current (IBa) were recorded by isometric tension recording and whole-cell patch clamp technique. Spermine, spermidine and putrescine inhibited spontaneous contraction of the gastric smooth muscle in a concentration-dependent manner. Spermine (2 mM) reduced high K+ (50 mM)-induced contraction to 16±6.4% of the control (n=9), and significantly inhibited IBa in a reversible manner (p<0.05; IC50=0.8 mM). Pre- and post-treatment of tissue with spermine (2-5 mM, n=10) also inhibited acetylcholine (10 µM)-induced phasic contraction to 5±6.4% of the control. Inhibitory effect of spermine on IBa was observed at a wide range of test potentials of current/voltage (I/V) relationship (p<0.05), and steady-state activation of IBa was shifted to the right by spermine (p<0.05). Spermidine and putrescine (1 mM each) also inhibited IBa to 51±5.7% and 81±5.3% of the control, respectively. And putrescine (1 mM) inhibited IBa at whole tested potentials (p<0.05) without significant change of kinetics (p<0.05). Finally, 5 mM putrescine also inhibited high K+-induced contraction to 53±7.1% of the control (n=4). These findings suggest that polyamines inhibit contractions of guinea-pig gastric smooth muscle via inhibition of VDCC.

Molecular cloning and functional expression of a VIP-specific receptor

Three receptors for VIP and pituitary adenylate cyclase-activating peptide (PACAP) have been cloned and characterized: PAC(1), with high affinity for PACAP, and VPAC(1) and VPAC(2) with equally high affinity for VIP and PACAP. The existence of a VIP-specific receptor (VIP(s)) in guinea pig (GP) teniae coli smooth muscle was previously surmised on the basis of functional studies, and its existence was confirmed by cloning of a partial NH(2)-terminal sequence. Here we report the cloning of the full-length cDNAs of two receptors, a VPAC(2) receptor from GP gastric smooth muscle and VIP(s) from GP teniae coli smooth muscle. The cDNA sequence of the VIP(s) encodes a 437-amino acid protein (M(r) 49,560) that possesses 87% similarity to VPAC(2) receptors in rat and mouse and differs from the VPAC(2) receptor in GP gastric smooth muscle by only two amino-acid residues, F(40)F(41) in lieu of L(40)L(41). In COS-1 cells transfected with the GP teniae coli smooth muscle receptor, only VIP bound with high affinity (IC(50) 1.4 nM) and stimulated cAMP formation with high potency (EC(50) 1 nM). In contrast, in COS-1 cells transfected with the GP gastric smooth muscle receptor, both VIP and PACAP bound with equally high affinity (IC(50) 2.3 nM) and stimulated cAMP with equally high potency (EC(50) 1.5 nM). We conclude that the receptor cloned from GP teniae coli smooth muscle is a VIP(s) distinct from VPAC(1) and VPAC(2) receptors. The ligand specificity in this species is determined by a pair of adjacent phenylalanine residues (L(40)L(41)) in the NH(2)-terminal ligand-binding domain.

Functional differences of Na+/Ca2+exchanger expression in Ca2+transport system of smooth muscle of guinea pig stomach

The plasma membrane ATP-dependent Ca2+ pump and the Na+/Ca2+ exchanger (NCX) are the major means of Ca2+ extrusion in smooth muscle. However, little is known regarding distribution and function of the NCX in guinea pig gastric smooth muscle. The expression pattern and distribution of NCX isoforms suggest a role as a regulator of Ca2+ transport in cells. Na+ pump inhibition and the consequent to removal of K+ caused gradual contraction in fundus. In contrast, the response was significantly less in antrum. Western blotting analysis revealed that NCX1 and NCX2 are the predominant NCX isoforms expressed in stomach, the former was expressed strongly in antrum, whereas the latter displayed greater expression in fundus. Isolated plasma membrane fractions derived from gastric fundus smooth muscle were also investigated to clarify the relationship between NCX protein expression and function. Na+-dependent Ca2+ uptake increased directly with Ca2+ concentration. Ca2+ uptake in Na+-loaded vesicles was markedly elevated in comparison with K+-loaded vesicles. Additionally, Ca2+ uptake by the Na+- or K+-loaded vesicles was substantially higher in the presence of A23187 than in its absence. The result can be explained based on the assumption that Na+ gradients facilitate downhill movement of Ca2+. Na+-dependent Ca2+ uptake was abolished by the monovalent cationic ionophore, monensin. NaCl enhanced Ca2+ efflux from vesicles, and this efflux was significantly inhibited by gramicidin. Results documented evidence that NCX2 isoform functionally contributes to Ca2+ extrusion and maintenance of contraction-relaxation cycle in gastric fundus smooth muscle.

Nonselective cation channels activated by the stimulation of muscarinic receptors in mammalian gastric smooth muscle.

Muscarinic receptors play key roles in the control of gastrointestinal smooth muscle activity. However, specific physiological functions of each subtype remain to be determined. Single cell RT-PCR experiments showed that all five subtypes of muscarinic receptors were present in circular smooth muscle cells of the guinea-pig gastric antrum. Nonselective cation channels (NSCC) activated by ACh or CCh are coupled to pertussis toxin (PTX)-sensitive Go protein through m4 subtype as well as m2 and m3 subtypes in guinea-pig stomach. CCh-activated currents (I(CCh)), especially the steady-state I-V relationship of I(CCh) showed a chracteristic U-shaped curve; reversal potential of around 0 mV and inward rectification at around +15 mV and a negative slope conductance at negative potential range. Under physiological conditions, the measured single channel conductance of NSCC was approximately 25 pS. The single channel conductance was modulated by external monovalent and divalent cations including Na+, Cs+, Li+, and Ca2+ through changing both the open probability and unitary conductance. Through the NSCC, Ca2+ can move into the cell from extracellular solution as well as Na+. Calculated fractional Ca2+ current of I(CCh) (f(Ca)) was around 1% at the 2 mM [Ca2+]o and at the 4 mM [Ca2+]o, f(Ca) was 2.3%. Quinidine blocked I(CCh) potently in a reversible manner; IC50 was 0.25 microM. There were two kinds of I(CCh) modulations through Ca(2+)-dependent pathways in guinea-pig gastric smooth muscle cells; 1) Facilitation of I(CCh) via Ca2+/CaM-dependent MLCK pathway, 2) Desensitization of I(CCh) via Ca(2+)-dependent PKC pathway. In the mouse stomach, all seven types of TRPC mRNA were detected with RT-PCR. On the basis of electrophysiological, pharmacological, and molecular biological experiments, we reported the mTRPC5 as a candidate for the NSCC activated by muscarinic stimulation in mouse stomach.

Muscarinic Receptors Controlling the Carbachol-Activated Nonselective Cationic Current in Guinea Pig Gastric Smooth Muscle Cells

Muscarinic receptor subtypes controlling the nonselective cationic current in response to carbachol (ICCh) were studied in circular smooth muscle cells of the guinea pig gastric antrum using putative muscarinic agonists and antagonists. Both oxotremorine-M (an M2-selective agonist) and CCh dose-dependently activated the cationic current with EC50 values of 0.21 ± 0.01 μm and 0.97 ± 0.06 μuM, respectively. In contrast, pilocarpine and McN-A 343 (an Mi-selective and a putative M4 agonist) were weak partial agonists. In response to 10 μM CCh, 4-DAMP, methoctramine and pirenzepine dose-dependently inhibited ICCh and had IC50 values of 1.91 ± 0.2 nM, 0.46 ± 0.07 μM and 8.33 ± 0.4 μM, respectively. 4-DAMP, methoctramine and pirenzepine shifted the concentration-response curves of ICCh to the right without significantly reducing the maximal current. Values of the apparent dissociation constant pA2 obtained from Schild plot analysis were 9.24, 7.72 and 6.62 for 4-DAMP, methoctramine and pirenzepine, respectively. Also, pertussis toxin completely blocked ICCh generation. These results suggest that the M2-subtype plays a crucial role in the activation of the ICCh and a block of the M3-subtype reduces the sensitivity of the M2-mediated response with no significant reduction of maximum response.

Suppression of the carbachol-activated nonselective cationic current by antibody against alpha subunit of Go protein in guinea-pig gastric myocytes.

In this study, we investigated which subtype of GTP-binding protein (G protein) is related to muscarinic activation of nonselective cation (NSC) channels in gastric smooth muscle. Inward cationic current was activated by the application of 50 microM carbachol (ICCh) at a holding potential of -60 mV with the same CsCl-rich solution in both pipette and bath. The same cationic current as ICCh was slowly activated by the dialysis of guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S]) through the pipette. Since it is known that pertussis toxin pretreatment can block ICCh, antibodies (Abs) against Galpha,i (anti-Galpha,i) or Galpha,o (anti-Galpha,o) were tested. Activation of ICCh was blocked by the addition of anti-Galpha,o. However, anti-Galpha,i Abs had no significant effect on ICCh. The expression of Galpha,o in guinea-pig gastric smooth muscle was confirmed by Western immunoblot analysis. These results suggest that Go-type protein may mediate signals from the muscarinic receptor to NSC channel in guinea-pig gastric myocytes.

CCK regulates nonselective cation channels in guinea pig gastric smooth muscle cells.

CCK has widespread effects in the gastrointestinal tract, stimulating pancreatic secretion and contraction of smooth muscles. The cellular mechanisms by which CCK causes smooth muscle contraction are poorly understood. We investigated the effects of CCK on guinea pig gastric smooth muscle cells using patch-clamp techniques. CCK caused contraction of cells accompanied by inward current. The conductance activated by CCK was nonselective for cations and showed little voltage dependence. Because ACh also activates nonselective cation current, we examined interactions between CCK and ACh. When CCK activated inward current, ACh caused no further effect. When CCK failed to activate current, subsequent ACh-activated current was larger and no longer exhibited its characteristic voltage dependence. Intracellular dialysis with guanosine 5'-O-(3-thiotriphosphate) caused similar changes in the voltage dependence of the ACh-activated current, suggesting a role for G proteins in regulation of the current. Activation of nonselective cation current would depolarize muscle and may contribute to the excitation mediated by CCK in tissues. These findings provide evidence that multiple types of receptors converge to regulate nonselective cation current.

Two types of stretch-activated channel activities in guinea-pig gastric smooth muscle cells.

Using cell-attached patch-clamp techniques, stretch-activated channels were investigated in guinea-pig gastric smooth muscle cells. Suction applied to the pipette interior or cell swelling induced by hypotonic bath solution induced two kinds of channel openings, a short-lasting and small opening (OS) and a long-lasting and large opening (OL) with conductances of 33 and 53 pS, respectively. Threshold suction for OS was lower than that for OL. In many patches examined, OS and OL coexisted. The distributions of open- and closed-times of OS could be fitted by the sum of two exponentials, the time constants of which were 1.3 and 9.3 ms for the open-time, and 0.6 and 8.4 ms for the closed-time. Both OS and OL were blocked by 100 microM Gd3+ in the pipette. The reversal potentials did not change upon altering [Cl-]o. The results indicate that OS and OL seem to represent non-selective cation channel activities, which do not discriminate among K+, Na+, and Ca2+. These channels may play an important role in the stretch-induced contraction in gastric muscle.

Characterization of beta-adrenoreceptors on smooth muscle cells from guinea pig stomach

To characterize the beta-adrenergic receptors on guinea pig gastric smooth muscle cells, we examined the effects of beta-adrenergic agonists and antagonists on biological activity, cellular adenosine 3',5'-cyclic monophosphate (cAMP), and radioligand binding. Adrenergic agonists, isoproterenol (ISO), epinephrine (EPI), and norepinephrine (NE), inhibited carbachol-stimulated contraction of muscle cells, with relative potencies (IC50S) of ISO (0.1 microM) greater than EPI (1.4 microM) greater than NE (11 microM). Each agonist increased cellular cAMP, with relative potencies (IC50S) of ISO (0.5 microM) greater than EPI (6.3 microM) greater than NE (56 microM). Binding of the nonselective beta-antagonist 125I-pindolol was temperature-dependent, saturable, reversible, and specific. 125I-pindolol binding was inhibited by the three agonists, with relative potencies (IC50S) of ISO (0.9 microM) greater than EPI (9.6 microM) greater than NE (112 microM). Pindolol inhibited binding of 125I-pindolol with an IC50 of 100 nM. The IC50 for inhibition of binding of 125I-pindolol by the relatively beta 2-selective antagonist ICI 118,551 was 70 nM and for the relatively beta 1-selective antagonist betaxolol was 1,000 nM. Computer analysis of the dose-inhibition curves for binding of 125I-pindolol for the antagonists indicated that gastric smooth muscle cells possess exclusively beta 2-adrenergic receptors of two classes, one class with a high affinity for ICI 118,551 (Kd = 50 nM) and the other with a low affinity for ICI 118,551 (Kd = 30 microM). Our results indicate that beta-adrenergic agonists interact with beta 2-adrenergic receptors on gastric smooth muscle cells to increase cellular cAMP and inhibit muscle contraction.

Isolation of guinea pig gastric smooth muscle cells.

Isolation of guinea pig gastric smooth muscle cells.

Motilin receptors on isolated gastric smooth muscle cells

Motilin has a stimulating effect on gastrointestinal motility. The mechanism of its action is not known. Direct and neuronal effects have been postulated. To determine if receptors are present on smooth muscle cells we investigated the effect of synthetic porcine motilin and its interaction with acetylcholine on isolated guinea pig gastric smooth muscle cells. Motilin elicited a dose-dependent contraction of gastric smooth muscle cells. Minimal (8.3 +/- 1.3%) and maximal (33.9 +/- 2.4%) responses were observed at 10(-12) and 10(-6) M, respectively. The ED50 of motilin was 10(-9) M. Acetylcholine also elicited a dose-response muscle contraction with a maximal response observed at 10(-7) M. Atropine (10(-7) M) completely inhibited the maximal response to acetylcholine but did not have any effect on the contractile response to motilin. In addition, dibutyryl guanosine 3',5'-cyclic monophosphate (10(-3) M) and substance P antagonist, spantide (10(-4) M), also did not inhibit the action of motilin. Acetylcholine (10(-11) M) shifted the dose-response curve of motilin to the left by 1.5 log units. The maximal response to the combination of motilin (10(-6) M) and acetylcholine (10(-11) M) was 32 +/- 3.2%, which was similar to the maximal response to motilin alone. It is concluded that distinct motilin and muscarinic receptors are present on guinea pig gastric smooth muscle cells. The interaction between motilin and acetylcholine is additive and not potentiative.