Dual concentration-dependent effects of phorbol 12, 13-dibutyrate on spontaneous and acetylcholine-induced electrical responses recorded from isolated circular smooth muscle of the guinea-pig stomach antrum.

Intracellular recordings of electrical activity were made from circular smooth muscle cells in small segments of tissue isolated from the guinea-pig stomach antrum. Every cell that was impaled exhibited a rhythmic generation of slow potentials. Experiments were carried out to test the effects of three different concentrations (1, 10 and 100 nM) of phorbol 12, 13-dibutyrate (PDBu) on these slow potentials and on the responses produced by acetylcholine (ACh), in the presence of nifedipine and N(omega)-nitro-L-arginine (nitroarginine), known inhibitors of L-type Ca-channels and nitric oxide synthase, respectively. The resting membrane potential was -62 +/- 7 mV, while the frequency and amplitude of the slow potentials were 1.6 +/- 0.1 cycle per min (cpm) and 33 +/- 1 mV, respectively. Application of 1 nM PDBu increased the frequency of slow potentials, with no significant change in the membrane potential and amplitude of slow potentials. At a concentration of 100 nM, PDBu depolarized the membrane by about 6 mV, and either decreased the amplitude and frequency of the slow potentials or abolished them. The amplitude and frequency of the slow potentials were not significantly changed in the presence of 10 nM PDBu. In the presence of chelerythrine (1-2 microM), a known inhibitor of protein kinase C (PKC), the increase in frequency of slow potentials by 1 nM PDBu and depolarization produced by 100 nM PDBu were not elicited. The increase in frequency of slow potentials by 100 nM ACh was inhibited by PDBu, in a concentration-dependent manner, and ACh-responses were abolished in the presence of 100 nM PDBu. These results indicate that PDBu has dual actions on the spontaneous activity of antral circular muscle, with low concentrations increasing and high concentrations inhibiting the frequency of the slow potentials. The former may be produced by activation of protein kinase C (PKC). As the ACh-induced excitation of slow potentials is inhibited by PDBu, a possible causal relationship between the inhibition and over-activation of PKC is considered.     

Binding of [3H]inositoltrisphosphate and [3H]phorbol 12,13-dibutyrate in rat hippocampus following transient global ischemia: a quantitative autoradiographic study

An in vitro quantitative autoradiographic binding study of the phosphatidylinositol system ligands [3H]inositol 1,4,5-trisphosphate (IP3) and [3H]phorbol 12,13-dibutyrate (PDBU) to rat brain slices was performed at 6, 12, 24, 28 and 72 h following a 20 min ischemic injury. PDBU binding showed a transient 20% decrease in the dentate gyrus and the CA3 the first 24 h as well as a 50% decrease in the CA1 at 72 h. A 50% decline in IP3 binding was seen in all regions at 6-12 h except the pyramidal cell layer of the CA1. This downregulation of calcium mobilizing intracellular receptors is probably a defence against ischemic neuronal cell death.     

Potassium flux in smooth muscle of frog stomach.

In frog stomach muscle fibers, normal steady-state K flux, estimated directly from 42K uptake, was 0.17 pmol/cm2 per s at 5 degrees C and 0.63 pmol/cm2 per s at 15 degrees C. Influx characteristics were studied at 5 degrees C, where backflux and diffusional delay effects are minimized. Steady-state K influx was a saturating function of external [K] over the range 0.25-11 mM [K]o; influx at normal and higher [K]o did not differ significantly. Na loading (in K-free or low K solution) strongly stimulated influx, which showed altered saturation kinetics; maximal K influx was a quasilinear function of internal [Na]. Ouabain (10(-4) M) reduced normal and stimulated K influx markedly. Ethacrynic acid (10(-3) M) caused net K loss and Na gain, but increased K influx fourfold; ouabain inhibited the stimulated influx by 50%. These results indicate that K influx depends mainly on cycling of the Na-K pump and is normally limited by Na efflux. Ethacrynic acid may stimulate another mode of pump operation, K-K exchange, and uncouple the normal operation.     

Phorbol-12,13-dibutyrate improves the quality of cytogenetic preparation in lymphoid malignancies

In cytogenetic preparation of lymphoid malignancies we investigated the quantitative and qualitative impact of phorbol-12,-13-dibutyrate (P) and of this tumor promoter in combination with the calcium ionophore A23187 (PA). Using parallel cultures of unstimulated and stimulated preparations, the effect was examined in 13 patients with malignant lymphomas and six patients with acute lymphoblastic leukemias (ALL). Focusing on high-quality analyzable metaphases, the best results were found in seven of 13 cases with lymphomas and five of six patients with ALL in the cultures supplemented with phorbol-12,13-dibutyrate. The yield of metaphases of good quality regarding length, spreading, and banding of chromosomes was regularly better in F-stimulated 24-hour culture (p < 0.05), followed by 48-hour cultures stimulated with P alone. Addition of the calcium-ionophore was of no further benefit. The yield of the unstimulated direct harvest was rather poor in nearly all patients investigated. Because no mutagenic effect of P was observed, the use of this mitogen may offer interesting perspectives in cytogenetic analysis of lymphoid malignancies and perhaps also in other tumors with low mitotic indexes.     

Postischemic binding of [3H]phorbol 12,13-dibutyrate and [3H]inositol 1,4,5-trisphosphate in the gerbil brain: an autoradiographic study.

Postischemic alteration of second messenger systems was investigated in the Mongolian gerbil, utilizing [3H]phorbol 12,13-dibutyrate and [3H]inositol 1,4,5-trisphosphate receptor autoradiography. Transient ischemia was induced for 10 min, and animals were allowed to survive for various recirculation periods of up to one month. [3H]Phorbol 12,13-dibutyrate binding in selectively vulnerable areas showed no significant change 1-24 h after ischemia except for a transient decline in a few regions. Thereafter, the binding in most of the selectively vulnerable areas showed significant alteration 48 h or seven days after ischemia. Interestingly, dentate molecular layer which was resistant to ischemia showed a significant elevation in the number of [3H]phorbol 12,13-dibutyrate binding sites. One month after ischemia, [3H]phorbol 12,13-dibutyrate binding showed significant reduction only in the striatum and the hippocampal CA1 sector where severe neuronal damage was seen morphologically. A significant elevation in the number of [3H]phorbol 12,13-dibutyrate binding sites was still seen in the dentate molecular layer one month after ischemia. In contrast, [3H]inositol 1,4,5-trisphosphate binding showed significant reduction in the selectively vulnerable regions 1-24 h after ischemia. Thereafter, [3H]inositol 1,4,5-trisphosphate binding in most of the selectively vulnerable areas markedly decreased up to one month after ischemia. In the dentate molecular layer, [3H]inositol 1,4,5-trisphosphate binding also showed significant reduction during recirculation except for a slight recovery 48 h and seven days after ischemia. One month after ischemia, the binding in all regions showed significant reduction. These results suggest that postischemic alteration of two second messenger (protein kinase C and inositol 1,4,5-trisphosphate) binding sites was produced with different processes in selectively vulnerable areas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol 12,13-dibutyrate enhances lateral redistribution of membrane glycoproteins in human blood lymphocytes

Nanomolar concentrations of 4 beta-phorbol 12,13-dibutyrate markedly enhanced the redistribution of concanavalin A receptors, the common leukocyte antigen and the Lyt-3 antigen in human blood lymphocytes, as measured by cap formation. The effect on the lateral mobility of these cell surface molecules was dose dependent and occurred within a few minutes of treatment. 12-O-Tetradecanoyl phorbol 13-acetate, another tumor promoter, was similarly active. 4 alpha-Phorbol 12,13-didecanoate, which does not have tumor-promoting activity, did not enhance cap formation. The effect of various drugs and treatments indicated that the phorbol ester-enhanced cap formation was energy and temperature dependent and required functional microfilaments. Retinoic acid, an antitumor-promoting agent, was inhibitory and trifluoperazine, an inhibitor of calmodulin-dependent processes, had a minor inhibitory effect. Protein secretion and synthesis, extracellular Ca2+/Mg2+ and functional microtubules did not seem to be involved. The enhanced capping was inhibited by the alkylating agents tosyl phenylalanyl chloromethyl ketone and tosyl lysyl chloromethyl ketone but not by other protease inhibitors. The effect of various amino acid derivatives suggested the participation of an esterase. A comparative study of dose response, kinetics and sensitivity to drugs indicated a direct correlation between the phorbol 12,13-dibutyrate-enhanced redistribution of membrane glycoconjugates and the phorbol ester-induced binding (adhesion) between human blood lymphocytes, a phenomenon previously described.     

Relationship between intrapulmonary airway diameter and smooth muscle tone in excised lungs.

1. Intrapulmonary bronchi in excised dog lungs were outlined with tantalum dust and stereoscopic radiographs taken during deflation and inflation of the lung after rinsing with solutions of saline, histamine, isoprenaline or EDTA. Dimensions of airways were calculated from measurements of the stereoscopic X-ray images. 2. After treatment with EDTA to minimize bronchial smooth muscle activity, airway diameters increased at all transpulmonary pressures (Ptp) and lung volumes relative to their diameter after treatment with histamine; airway hysteresis in relation to Ptp decreased. 3. At low lung volumes, the per cent increase from histamine to EDTA for airways of different sizes was the same (24-30%) but at high volumes (30 cm H2O distending pressure) the dilatation induced by EDTA was 30% for airways less than 3.0 mm diameter and 13% for those greater than 5.0 mm diameter. 4. Even at high lung volumes, intrapulmonary airways are free to constrict or dilate in spite of the stiffness of the supporting parenchyma.     

Ionic fluxes in rat uterine smooth muscle.

1. The K+, Na+ and Cl-fluxes from the oestrogen-stimulated rat uterine smooth muscle were measured using radioactive tracers. 2. The cellular compartment contained a concentration of K+ of 173-6 mM which exchanged at a rate of 5-9 x 10(-12) mol.cm-2.sec-1. 3. Cl- exchanged at a rate of 6-9 x 10(-12) mol.cm-2.sec-1 from a cellular compartment having a concentration of 39-3 mM. 4. The methods used for the evaluation of Na+ movements over-estimate both influx and efflux values. If an average value of flux of 9-2 x 10(-12) mol.cm-2.sec-1 is considered we obtain PNa+/PK+ ratios of 2-4 (-42 mV) or 1-3 (-57-6 mV), which are too high and do not correspond to electrophysiological evidence. 5. The relative permeabilities PCl-/PK+ in the case of a membrane potential of -42-0 mV could be 0-8, or 1-3 in the case of a membrane potential of -57-6 mV. 6. Both conductances GK+ and GCl- seem to play an important role in determining membrane conductance.     

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. Received: 10 August 1995/Received after revision: 9 November 1995/Accepted: 10 November 1995     

Spike-free activation mechanism in smooth muscle of guinea-pig stomach

Electrical and mechanical activity of circular muscle strips of guinea-pigs stomach, taken from the distal corpus/proximal antrum region, were recorded. Spontaneous activity consisting of phasic contractions combined with bursts of spike potentials was suppressed by verapamil (5-10-6 - 2-10-5 mol/l). Under these conditions acetylcholine produced a spike-free tonic activation. Under normal conditions phasic contractions were superimposed on this tonic activation. The acetylcholine-induced activation, therefore, consists of two different components, one of which can be selectively blocked with verapamil. Both components disappear quickly in calcium-free solution. It can be concluded that two different calcium activation systems are responsible for the two components of activation. In comparative studies with taenia coli preparations a comparable spike-free tonic activation was not found.     

Effect of lithium of responsiveness of rat uterine smooth muscle to angiotensin.

Studies were carried out to investigate the effect of Na+ deprivation on the response of rat uterine smooth muscle to angiotensin II (AII). Replacement of Na+ with Li+ in a low-Ca2+-, Mg2+-free solution resulted in a concentration-dependent inhibition of both AII and acetylcholine (ACh)-induced contractions. Inhibition was noted at 10% Li+ substitution and was usually complete when 50% of the Na+ was replaced by Li+. The inhibition was not observed when an equivalent amount of tris(hyroxymethyl)aminomethand (Tris+) or sucrose was used to replace Na+. However, elevation of Ca2+ concentration from 0.2 to 0.5 mM did prevent full expression of the Li+ inhibition of AII. The Li+ effect was rapid in onset (60% inhibition after 5 min preincubation) and readily reversible following removal of the Li+. If, however, the tissue was placed in a depolarizing solution (151 mM KCL), Li+ inhibition of AII WAS NO LONGER OBSERVED. These results are interpreted to mean that Li+ interferes with an essential Ca2+-dependent step involved in membrane excitation following agonist-receptor interaction. The results are discussed in relation to a previously suggested model for AII-induced contractions in this tissue.     

Potential regulatory role of inflammatory cells on local vascular smooth muscle tone

A neutrophil-derived relaxing factor (NDRF) from oyster glycogen (OG)-elicited rat PMN, which causes an endothelium independent relaxation of rat aorta, and which is pharmacologically indistinguishable from endothelium-derived relaxing factor (EDRF) has been described [1]. Experiments were designed to evaluate the presence of NDRF in PMN from rat-whole blood,-carrageenan pleurisy.,-OG peritonitis, and guinea pig (GP)-OG peritonitis, as well as in OG-elicited rat macrophages (MØ). Significant vascular relaxing activity was found using rat PMN from OG peritonitis and carrageenen pleurisy, as well as from OG-MØ. Little or no activity was found in rat whole blood PMN or PMN from GO-OG peritonitis. These results suggest that NDRF activity may be expressed upon cellular migration to an inflammatory site in the rat, and may not be present in all species. Also, all inflammatory cells examined were capable of reversing EDRF-dependent relaxations when stimulated to produce superoxide anion suggesting a dual regulatory role for these cells on local vascular tone.     

Effect of dopamine on smooth muscles of the rat stomach and characteristics of α adrenoreceptors of muscle cells of the gastrointestinal tract

Dopamine and phenylephrine lower the tone of the smooth muscles of isolated strips of rat stomach in concentrations of 10^–6M or more. The concentration-effect curves have the same slope. The effect of dopamine is unchanged in the presence of propranolol (5·10^–6 g). Phentolamine (and also dihydroergotamine and tropaphen) exhibits equal antagonism to phenylephrine and dopamine. It is concluded that dopamine and phenylephrine relax the smooth muscles of the stomach by their action on adrenoreceptors. The latter differ from the adrenoreceptors of the vas deferens (rats) in their high sensitivity to the blocking effect of certain neuroleptics, namely haloperidol, trifluoroperazine, and chlorpromazine, pA₂ for which (8.11–8.64) is of the same order as pA₂ for adrenolytics (7.76–8.46). The similarity and difference between adrenoreceptors of muscles of the gastrointestinal tract and inhibitory dopaminergic receptors of nerve cells are discussed.Department of Pharmacology, A. M. Gor'kii Medical Institute, Donetsk. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Zakusov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 2, pp. 180–182, February, 1978.