Direct contractile effect of motilin on isolated smooth muscle cells of guinea pig small intestine.

We examined the direct effect of motilin on longitudinal and circular smooth muscle cells isolated from the guinea pig small intestine. In addition, the effects of 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxy-benzoate hydrochloride (TMB-8, an inhibitor of intracellular Ca(2+)-release), verapamil (a voltage-dependent Ca(2+)-channel blocker), and removal of extracellular Ca2+ were investigated to evaluate the role of intracellular Ca2+ stores and extracellular Ca2+ on the muscle contraction induced by motilin. The effects of atropine (a muscarinic receptor antagonist), spantide (a substance P receptor antagonist) and loxiglumide (a CCK-receptor antagonist) were also examined to determine whether the motilin-induced contraction was independent of those receptors. Motilin induced a contraction of the longitudinal and circular smooth muscle cells in a dose-dependent manner with the maximal effect attained after 30 seconds of incubation. The ED50 values were 0.3 nM and 0.05 nM, respectively. TMB-8 suppressed completely the motilin-induced contraction of both types of smooth muscle cells. Verapamil had only a slight suppressive effect. Removal of extracellular Ca2+ did not have any significant influence on motilin-induced contraction. The contractile response to motilin was not affected by atropine, spantide or loxiglumide. Our findings showed that:1) motilin has a direct contractile effect on both longitudinal and circular smooth muscle cells; 2) this contractile effect is not evoked via muscarinic, substance P or CCK receptors, and 3) the intracellular release of Ca2+ plays an important role in the contractile response to motilin on both types of smooth muscle cells.     

Noncholinergic contractile response to nicotine in the guinea pig isolated tracheal smooth muscle

The existence of substance P immunoreactive nerves in the trachea of guinea pig is known. In this study, capsaicin induced a long-lasting and marked contraction in the guinea pig trachea and nicotine-induced contraction was partially reduced in the capsaicin-treated muscle. Furthermore, the contractile response to nicotine (10(-5) M) in the presence of atropine (10(-7) M) was abolished by a substance P antagonist, [D-Arg1, D-Pro2, D-Trp7,9 Leu11]substance P (10(-5) M). These findings suggest that noncholinergic contractile response to nicotine may be due to the release of material(s) resembling substance P in the isolated tracheal smooth muscle preparation of guinea pig.     

Desensitization of isolated smooth muscle cells from guinea pig taenia caecum to acetylcholine

The role of tissue organization of smooth muscle in short-term desensitization to acetylcholine (ACh) was examined by studying the desensitization of isolated single cells from guinea pig taenia caecum. Cells were isolated by collagenase digestion. The conditions during cell isolation were adjusted to obtain cells that showed repeated contractions. The cells contracted on treatment with 10(-7)-10(-6) M ACh, showing an all-or-none response. Desensitized cells also showed an all-or-none response but required a higher concentration of ACh for induction of contraction; i.e., the magnitude of their maximal response was not changed appreciably but the threshold concentration of ACh for their contraction was raised. Incubation of the whole tissue with 10(-4) M ACh for 10 min also caused desensitization. This desensitization was accompanied by reduction of the contractile response at intermediate concentrations. The mode of desensitization of isolated cells determined from the average response of the isolated cells was almost the same as that of whole muscle. It is concluded that the desensitization occurred in each cell irrespective of its tissue organization and that the desensitization was due to an increase of the threshold for contraction to ACh of each cell.     

Atp-activated ionic permeability in smooth muscle cells isolated from the guinea pig urinary bladder

Smooth muscle cells from the guinea pig urinary bladder were investigated by voltage clamping at the plasma membrane and using an intracellular perfusion technique. Applying adenosine triphosphate (ATP) at a concentration greater than 3 × 10^–8 M and at a membrane potential of –100 to –30 mV produced a rise in fast inward transmembrane current. A similar effect was exerted by adenosine diphosphate (ADP) and -, -, and ,-methylene ATP. Application of guanosine triphosphate, inosine triphosphate, adenosine monophosphate (AMP), and adenosine failed to activate this current. It was found that AMP blocks ATP receptors competitively. No pharmacological differences were found between the latter ATP receptors and those of rat sensory neurons. The ATP receptors were rapidly desensitized and recovered their sensitivity to agonists extremely slowly. Speed of desensitization was reduced by a decrease in ATP concentration.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 1, pp. 95–100, January–February, 1987.     

Dual effect of bombesin and gastrin releasing peptide on gastric emptying in conscious cats

The effect of bombesin (BBS) and gastrin releasing peptide (GRP) on gastric emptying was studied in conscious cats. This effect was measured simultaneously with antral motility. Acid and pepsin secretions as well as blood hormonal peptide release were additionally measured. A dual effect was observed. First, BBS and GRP slowed gastric emptying of liquids, while antral motility was decreased, then after 60 minutes of continuous intravenous infusion, antral motility returned to basal values and gastric emptying effect reversed. The mechanism of this peculiar action is independent of gastrin, pancreatic polypeptide, somatostatin and motilin release and most probably connected with a cholinergic stimulation induced by the peptides, the late predominance of which counterbalances the inhibitory effect of bombesin-like peptides on antral motility.     

Depressing action of nitroglycerin on voltage-activated calcium current in isolated smooth muscle cells of the guinea pig gut

The effect of nitroglycerin (NG) on inward voltage-activated calcium current (I _Ca) was studied in isolated smooth muscle cells (SMC) of the guinea pigtaenia coli with the voltage clamp technique in an intracellular dialysis mode. Addition of NG (10^–7 to 10^–4 M) to the extracellular solution reduced theI _Ca amplitude and increased theI _Ca inactivation rate. The maximum inhibition ofI _Ca (on the average, by 41.7 ± 4.8%,n=13) was produced by 10^–4 M NG; the inhibition was dose-dependent. No shift of theI _Ca current-voltage curve under the NG influence was observed. Application of dibutyril-cGMP (2·10^–4 M), a membrane-penetrating analog of cyclic 3,5-guanosine monophosphate (cGMP), likewise decreased theI _Ca amplitude and increased its inactivation rate. The results obtained suggest that the NG inhibitory effect onI _Ca is related to a cGMP-dependent modulation of the voltage-activated calcium channels of L-type in the SMC membrane in the guinea pigtaenia coli.Neirofiziologiya/Neurophysiology, Vol. 26, No. 3, pp. 218–222, May–June, 1994.     

Direct effect of complement factor C5a on the contractile state of isolated smooth muscle cells

The complement-(C) derived factor C5a has long been recognized as a potent contractile agonist in smooth muscle (1,2); however, controversy remains as to whether the effects of this anaphylatoxin are direct or secondary to the release of histamine (3) and/or other mediators (4-8) from nonmuscle cells within the tissue. To resolve this controversy, we have assessed the contractile effects of purified human C5a and C5a des Arg in a homogeneous preparation of enzymatically dispersed smooth muscle cells derived from the stomach of the toad, Bufo marinus. This preparation, which is insensitive to histamine at concentrations as high as 10(-4) M, responds normally to a variety of electrical (9), mechanical (10), and pharmacologic (11, 12) stimuli. These smooth muscle cells also respond to purified human anaphylatoxin; exposure to the cells to purified human C5a or C5a des Arg produce contractions of the smooth muscle cells that are accompanied by increased Ca2+ influx. The contractile response was unaffected by antagonists to histamine or acetylcholine but was reduced by 30% by pretreatment with the leukotriene antagonist FPL55712. A direct contractile effect of C5a on amphibian smooth muscle cells is suggested.     

Effects of guinea pig vasoactive intestinal peptide on the isolated perfused guinea pig heart

The pharmacological effects of guinea pig vasoactive intestinal peptide (VIP) were studied in isolated perfused guinea pig hearts. Bolus injections of VIP produced a dose-dependent tachycardia that was not affected by atenolol. A decrease in amplitude of ventricular contractions occurred in response to all doses of VIP. This response was preceded by a small increase in amplitude in 3 of 6 hearts at the highest dose. VIP produced a decrease in perfusion pressure which was prominent after coronary tone was elevated with [Arg8]-vasopressin. The present findings support speculation that VIP may have a role in the regulation of heart rate and coronary blood flow.     

Calcium waves in intact guinea pig gallbladder smooth muscle cells

Intracellular Ca(2+) waves and spontaneous transient depolarizations were investigated in gallbladder smooth muscle (GBSM) whole mount preparations with intact mucosal layer [full thickness (FT)] by laser confocal imaging of intracellular Ca(2+) and voltage recordings with microelectrodes, respectively. Spontaneous Ca(2+) waves arose most often near the center, but sometimes from the extremities, of GBSM cells. They propagated regeneratively by Ca(2+)-induced Ca(2+) release involving inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] receptors and were not affected by TTX and atropine (ATS). Spontaneous Ca(2+) waves and spontaneous transient depolarizations were more prevalent in FT than in isolated muscularis layer preparations and occurred with similar pattern in GBSM bundles. Ca(2+) waves were abolished by the Ins(1,4,5)P(3) receptor inhibitors 2-aminoethoxydiphenyl borate and xestospongin C and by caffeine and cyclopiazonic acid. These events were reduced by voltage-dependent calcium channels (VDCCs) inhibitors diltiazem and nifedipine, by PLC inhibitor U-73122, and by thapsigargin and ryanodine. ACh, CCK, and carbachol augmented Ca(2+) waves and induced Ca(2+) flashes. The actions of these agonists were inhibited by U-73122. These results indicate that in GBSM, discharge and propagation of Ca(2+) waves depend on sarco(endo)plasmic reticulum (SR) Ca(2+) release via Ins(1,4,5)P(3) receptors, PLC activity, Ca(2+) influx via VDCCs, and SR Ca(2+) concentration. Neurohormonal enhancement of GBSM excitability involves PLC-dependent augmentation and synchronization of SR Ca(2+) release via Ins(1,4,5)P(3) receptors. Ca(2+) waves likely reflect the activity of a fundamental unit of spontaneous activity and play an important role in the excitability of GBSM.     

Effect of calmidazolium (R 24571) on the electrical and contractile properties of smooth muscle cells in the guinea pig ureter

Calmidazolium in macromolecular concentrations inhibited the electric and contractile activity of smooth muscle cells (SMC). The concentrations causing a 50% inhibition of oscillations on the action potential (AP) plate were equal to 1 X 10(-6) microM, AP amplitude was 3 X 10(-6) microM and contraction amplitude was 1 X 10(-6) microM. Calcium ionophore A 23187/8 X 10(-7) microM, added to the normal Krebs solution, decreased rapid AP components amplitude and increased the contraction power of the isolated SMC strip by 62 +/- 9%. A 23187, though to a lesser extent, increased smooth muscle contractions during the action of calmidazolium. With combined use of A 23187 and calmidazolium, rapid AP components were depressed to a greater extent than each of them taken separately. The data obtained point to the presence of calmodulin or similar protein in SMC of the calcium channels.     

Identification of gastrin releasing peptide-related substances in guinea pig and rat brain

Rat and guinea pig brain extracts were examined for the occurrence of gastrin-releasing peptide (GRP)-like substances by sequence specific radioimmunoassays interfaced with gel filtration and reversed phase high performance liquid chromatography (RP-HPLC). Tryptic digestion of the immunoreactive peptides followed by RP-HPLC was used to further characterize GRP-related peptides in brain. Using these analytical techniques it was found that guinea pig brain extracts contained a peptide with characteristics identical to authentic GRP (27 amino acid residues long). A carboxyterminal fragment with the characteristics of GRP(18–27) as well as a respective aminoterminal fragment with the characteristics of GRP(1–16) were also present in guinea pig brain extracts. The GRP(18–27) seems to correspond to the bombesin related material that has been described previously in mammalian brain extracts.Rat brain extracts also contained a peptide with the characteristics of GRP(18–27). The corresponding aminoterminal fragment, however, behaved differently on RP-HPLC from authentic GRP(1–16) and it was not recognized by antibodies directed to the aminoterminal tridecapeptide fragment of authentic GRP. Similarly the GRP-like peptide from rat brain did not comigrate on RP-HPLC with authentic GRP and was unreactive to antibodies directed toward the aminoterminus of GRP.     

Receptor type for cholecystokinin on isolated intestinal muscle cells of the guinea pig

Smooth muscle cells isolated from the longitudinal muscle layer of guinea pig ileum were used to determine the presence and type of cholecystokinin/gastrin receptor mediating contraction. This was accomplished with a series of cholecystokinin and gastrin agonists (CCK-8, des(SO3)CCK-8, gastrin-17, des(SO3)gastrin-17 and pentagastrin) and antagonists (glutaramic acid derivatives CR 1392, CR 1409, CR 1505 and proglumide). The order of potency of agonists based on EC50 values derived from concentration-response curves was: CCK-8 greater than des(SO3)CCK-8 greater than gastrin-17 greater than des(SO3)gastrin-17. The inhibitory dissociation constant (Ki) for the antagonist CR 1505 derived from Schild plots was the same whether sulfated CCK-8 or desulfated gastrin-17 was used as agonist (4.47 +/- 0.76 versus 4.68 +/- 0.78 nM). Pentagastrin acted as a partial agonist and inhibited partially the response to CCK-8. The Ki values determined for all antagonists with pentagastrin as agonist were similar to those with CCK-8 as agonist. The order of potency of agonists and the independence of Ki values from the type of agonist used implied that CCK and gastrin interact with one receptor type; the receptor is more sensitive to CCK-8 but is minimally influenced by sulfation of the tyrosine residue. In this respect, the receptor appears to be distinct from the CCK receptor on gallbladder muscle cells and pancreatic acinar cells.     

Relaxant activity of atriopeptins in isolated guinea pig airway and vascular smooth muscle

Atriopeptins are circulating peptide hormones which are secreted by atrial tissue and act at the kidney. Because the atriopeptins survive passage through the pulmonary circulation, they also may be involved in the modulation of airway or pulmonary vascular smooth muscle tone. Using in vitro organ bath techniques, atriopeptins were found to induce potent concentration-dependent relaxation of isolated guinea pig trachea, and pulmonary artery with a rank order of potency: atriopeptin III greater than atriopeptin II greater than atriopeptin I. Atriopeptin-induced smooth muscle relaxation was observed to be a direct response since it was not mediated by activation of relaxant VIP receptors, beta-adrenergic receptors, or H2 receptors nor affected by cyclooxygenase inhibition or denuding of the vasculature or trachea of endothelial and epithelial cells. The time course of atriopeptin II-induced relaxation of the pulmonary artery was transient in contrast to the prolonged relaxations on the trachea. The transient relaxant responses of atriopeptin II on pulmonary artery were not due to metabolism of atriopeptin II to atriopeptin I by angiotensin-converting enzyme since pretreatment with captopril did not augment the response. These results seem to indicate that distinct atriopeptin receptors may exist in airway and pulmonary arterial smooth muscle and that activation of these relaxant receptors may play an important role in the regulation of pulmonary vascular and bronchomotor tone.     

Distribution of contractile proteins in single isolated smooth muscle cells from the ascidian body-wall muscle

1. Relaxed cells isolated from ascidian body-wall muscle were morphologically very similar to relaxed common smooth muscle cells. 2. The contracted cells, however, possessed striations which were resolved into a repeating pattern of light and dark bands using phase contrast microscope. 3. The relaxed ascidian cells treated with Triton X-100 were contracted and showed the striations by adding Ca2+. 4. By an indirect immunofluorescence method, it was clearly seen that antiactin spread uniformly in the relaxed cells, while this antibody was concentrated on the dark bands of striations in the contracted cells.     

Inhibitory synaptic potentials of the smooth muscle cells of guinea pig taenia coli

A single submaximal intramural application of rectangular stimuli (duration 0.2–0.5 msec) to an atropine-treated taenia coli muscle band evoked inhibitory postsynaptic potentials (IPSP) and a marked relaxation of the muscle band in the vast majority of muscle cells. The latency period of the IPSP was 122±16 msec; the times for a rise and fall of amplitude were 96±8 and 370±60 msec, respectively. The mean latency period of muscle relaxation was 800 msec. The latency period, and especially the amplitude of the IPSP depended on the intensity of the intramural stimulation. This indicates that one muscle cell is inhibited by several nerve fibers. IPSP evoked by threshold stimuli displayed a tendency toward summation, while the amplitude of the second and of subsequent IPSP evoked by low-frequency maximal stimuli was always less than that of the first IPSP. After periodic stimulation (frequency 10–60 impulses/min) was discontinued, a posttetanic decrease in IPSP amplitude was observed. Anodic polarization of the muscle band with a direct current raised the effectiveness of synaptic transmission, as was evidenced by the considerable increase in IPSP amplitude. When the muscle membrane was hyperpolarized with noradrenaline, IPSP inhibition was reversible. This is evidence that the unknown mediator and noradrenaline have a common ionic inhibitory mechanism.A. A. Bogomol'ts Institute of Physiology of the Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 2, No. 5, pp. 544–551, September–October, 1970.     

Inward current in single smooth muscle cells of the guinea pig taenia coli

Using the tight-seal voltage-clamp method, the ionic currents in the enzymatically dispersed single smooth muscle cells of the guinea pig taenia coli have been studied. In a physiological medium containing 3 mM Ca2+, the cells are gently tapering spindles, averaging 201 (length) x 8 microns (largest diameter in center of cell), with a volume of 5 pl. The average cell capacitance is 50 pF, and the specific membrane capacitance 1.15 microF/cm2. The input impedance of the resting cell is 1-2 G omega. Spatially uniform voltage-control prevails after the first 400 microseconds. There is much overlap of the inward and outward currents, but the inward current can be isolated by applying Cs+ internally to block all potassium currents. The inward current is carried by Ca2+. Activation begins at approximately -30 mV, maximum ICa occurs at +10-+20 mV, and the reversal potential is approximately +75 mV. The Ca2+ channel is permeable to Sr2+ and Ba2+, and to Cs+ moving outwards, but not to Na+ moving inwards. Activation and deactivation are very rapid at approximately 33 degrees C, with time-constants of less than 1 ms. Inactivation has a complex time course, resolvable into three exponential components, with average time constants (at 0 mV) of 7, 45, and 400 ms, which are affected differently by voltage. Steady-state inactivation is half-maximal at -30 mV for all components combined, but -36 mV for the fast component and -26 and -23 mV for the other two components. The presence of multiple forms of Ca2+ channel is inferred from the inactivation characteristics, not from activation properties. Recovery of the fast channel occurs with a time-constant of 72 ms (at +10 mV). Ca2+ influx during an action potential can transfer approximately 9 pC of charge, which could elevate intracellular Ca2+ concentration adequately for various physiological functions.     

Outward current in single smooth muscle cells of the guinea pig taenia coli

In single myocytes of the guinea pig taenia coli, dispersed by enzymatic digestion, the late outward current is carried by K+. It has both a Ca2+-activated component and a voltage-dependent component which is resistant to external Co2+. The reversal potential is -84 mV, and the channel(s) for it are highly selective to K+. At 33 degrees C, the activation follows n2 kinetics, with a voltage-dependent time constant of 10.6 ms at 0 mV, which shortens to 1.7 ms at +70 mV. Deactivation follows a single-exponential time course, with a voltage-dependent time constant of 11 ms at -50 mV, which lengthens to 33 ms at -20 mV. During a 4.5-s maintained depolarization, IK inactivates, most of it into two exponential components, but there is a small noninactivating residue. It is surmised that during an action potential under physiological conditions, there is sufficient IK to cause repolarization.     

Calcium antagonists and contractile responses in rat vas deferens and guinea pig ileal smooth muscle.

The effect of depletion of extracellular Ca2+ (Ca2+ext) on the loss of responsiveness of the guinea pig ileal longitudinal muscle (g.p.i.l.m.) and the rat vas deferens (r.v.d.) to K+ and cis-2-methyl-4-dimethylaminomethyl-1,3-dioxolane methiodide (CD), and K+ and noradrenaline (NA), has been examined and compared with the effects of a variety of local anesthetics and calcium antagonists. The results indicate that qualitative similarities are apparent with respect to the dependence of agonist-induced activity on Ca2+ext in both the g.p.i.l.m. and r.v.d. Distinct differences, however, in the Ca2+ translocation processes in these two tissues, in response to the different agonists, can be shown by the use of a variety of 'calcium antagonists' thus indicating that such translocation processes are both tissue and agonist selective. It is thus noted that, contrary to the Ca2+ depletion studies, D 600 and the usually more potent BAY-1040 showed no discrimination of action or potency in their ability to inhibit components of the NA response in the r.v.d. In contrast, D 600 and the more potent BAY-1040 selectively inhibited the tonic component of the K+ response. Treatment with SKF 525A and parethoxycaine (PC) in the g.p.i.l.m. and SKF 525A in the r.v.d. resulted in a nonselective inhibition of responses of the tissues to all stimulants. However, in the r.v.d. PC potentiated NA action, and its methobromide (MeBr) derivative potentiated both NA and K+ action and also, like PC, partially shifted to the left the dose-response curve to Ca2+ in NA-depolarizing Ca-free Tyrode's. The quaternary MeBr and the tertiary 2-chloroethyl (2Cl) derivatives of SKF 525A and PC were selectively more effective against CD- than K+ supported contractile activity in the g.p.i.l.m. and the 2Cl derivatives were more effective against NA than K+ responses in the r.v.d. The 2Cl derivative of PC also was more effective in antagonizing the Ca2+ dose-response curve in high-CD or high-NA than in high-K+ Ca2+-free Tyrode's.