Tonic Component Of Contraction Research Articles

Shortening velocities of rat mesenteric lymphatics during spontaneous and agonist‐induced contractions

Determination of the maximum shortening velocity (Vmax) for lymphatic smooth muscle is critical to the analysis of lymphatic contractile properties. Rat mesenteric lymphatics were subjected to isotonic release protocols during stimulated or spontaneous contractions using a modified wire myograph. Maximal activation was evoked using Substance P (SP) and K+. Upon activation at optimal preload, lymphatics exhibited time-dependent force (F) development, with peak active F = 0.34±0.02 mN and subsequent lower plateau F = 0.21±0.02 mN at ~90 sec. At the peak, Vmax = 0.48±0.04 lengths/sec, whereas Vmax at the plateau declined to 0.26±0.03 length/sec. In cannulated lymphatics under isobaric conditions, shortening velocity (V) was estimated from dD/dt during spontaneous contractions. Paradoxically, V was ~10-fold higher than in maximally activated vessels. Further, V declined with progressive activation by SP. Using the myograph, releases were then triggered at a predetermined F during the upstroke of spontaneous isometric contractions. Vmax so obtained was in closer agreement with isobaric V. The results suggest that lymphatic activation is associated with a profound slowing of V and are consistent with lymphatic smooth muscle being a biochemical hybrid of smooth and striated muscle that exhibits different mechanical properties in the phasic and tonic components of contraction. Supported by NIH HL-075199.

Mechanism of activation of the tonic component of contraction in myocytes of guinea pig heart.

Contractions of myocytes of guinea pig heart consist of a phasic component relaxing independently on the voltage and a tonic component relaxing upon repolarization. We found previously that Ca(2+) activating the tonic component is released from the sarcoplasmic reticulum. In this study, we analysed the mechanism of activation and maintenance of this release. Experiments were performed at 37 degrees C in ventricular myocytes of guinea pig heart. Voltage-clamped myocytes were stimulated by the pulses of the duration of 300 ms to 15-45 s from the holding potential of -40 to +5 mV. [Ca(2+)](i) was monitored as fluorescence of Indo-1 and contractions were recorded with the TV edge-tracking system. Myocytes responded to the short and long pulses with phasic contraction or Ca(2+) transient followed by the sustained contraction or increase in [Ca(2+)](i). Repolarization brought about relaxation. 10 mmol L(-1) Ni(2+) blocking Na(+)/Ca(2+) exchange superfused during the tonic component increased its amplitude. Superfusion of Ca(2+)-free solution during sustained contraction brought about relaxation both in normal cells and in cells superfused with Ni(2+) despite preserved sarcoplasmic reticulum Ca(2+) content assessed with caffeine spritz. Relaxing effect of Ca(2+)-free solution was not affected by carboxyeosin, a blocker of sarcolemmal Ca(2+)-ATPase. Tonic component of contraction and of Ca(2+) transient was inhibited by 200 micromol L(-1) ryanodine, a blocker of Ca(2+) release channels of the sarcoplasmic reticulum and by 20 micromol L(-1) nifedipine, a blocker of L-type I(Ca). Tonic component of contraction results from Ca(2+) release via the sarcoplasmic reticulum Ca(2+) channels activated by sustained, nifedipine-sensitive and Ni(2+)-insensitive Ca(2+) influx. Alternatively, the SR Ca(2+) release is activated by voltage, the dihydropyridine receptors acting like voltage sensors.

The effect of progesterone on spontaneous and agonist-evoked contractions of the rat aorta and portal vein.

The mechanisms underlying the suppression of vasocontractility caused by progesterone were investigated by studying changes in the contractile force of rat isolated aorta and portal vein, induced by altering extracellular concentrations of noradrenaline (NA) potassium ions (K+) and calcium ions (Ca2+). In the aorta, progesterone (10 microM) had a general suppressive effect on NA-, Ca2+- and K+-induced contractions. In contrast, in the portal vein a more selective suppression of contractions was observed. Both tonic and phasic components of contractions induced by cumulative addition of Ca2+ to tissues equilibrated in Ca2+-free saline were suppressed. The phasic but not tonic components of contractions induced by NA addition were suppressed. There was no significant effect on tonic contractions induced by elevated (40-120 mM) K+, but a concentration-dependent suppression of the phasic component of contractions was observed during depolarisation with smaller elevations of K+ concentrations (5-20 mM). These results suggest that on the portal vein the suppressive effect of progesterone is due to a potassium channel opening action, whilst on the aorta a different or additional mechanism of suppression exists.

Effect of niflumic acid on electromechanical coupling by tachykinin NK1 receptor activation in rabbit colon

We have investigated the effect of the Cl− channel blocker, niflumic acid, on the contractile response and electromechanical coupling activated by stimulation of the tachykinin NK1 receptor in the longitudinal muscle of rabbit proximal colon, in the presence of indomethacin (5 μM). The application of submaximal equieffective concentrations of the tachykinin NK1 receptor-selective agonist [Sar9]substance P sulfone (30 nM), of carbachol (300 nM) and KCl (40 mM0, produced distinct phasic and tonic components of contraction. Niflumic acid (10–100 μM) preferentially and markedly inhibited the tonic component of the response to [Sar9]substance P sulfone and to carbachol, without affecting the response to KCl. Nifedipine (1 μM) abolished the response to KCl and greatly reduced the response to [Sar9]substance P sulfone and carbachol. The nifedipine-resistant response to [Sar9]substance P sulfone was attenuated by niflumic acid (100 μM), while that to carbachol was unaffected. In sucrose gap experiments, superfusion with niflumic acid (100 μM), in the presence of nifedipine (3 μM), produced membrane hyperpolarization, which was totally blocked by tetraethylammonium (10 mM). Niflumic acid inhibited both depolarization and contraction induced by [Sar9]substance P sulfone, both in the absence or in the presence of tetraethylammonium. The present findings support the idea that a niflumic acid-sensitive mechanism, probably an effect on Cl− channels, takes part in the post-receptorial events activated by tachykinin NK1 receptor stimulation in the longitudinal muscle of rabbit colon, and suggest that this mechanism would be more important for generating the sustained tonic than the phasic component of contraction.

Specialization of tachykinin NK 1 and NK 2 receptors in producing fast and slow atropine-resistant neurotransmission to the circular muscle of the guinea-pig colon

We studied the relative contribution of tachykinin NK 1 and NK 2 receptors in producing nonadrenergic noncholinergic excitation of the circular muscle of the guinea-pig proximal colon in response to electrical field stimulation. All experiments were performed in the presence of atropine, guanethidine, indomethacin, apamin and l-nitroarginine. In organ bath experiments, electrical stimulation produced a tetrodotoxin-sensitive frequency-dependent contraction. The NK 1 receptor antagonists, FK 888 (1–10 μM) and GR 82,334 (0.3–3 μM) markedly reduced but did not abolish the nonadrenergic noncholinergic response. The NK 2 receptor antagonist, GR 94,800 (0.3–3 μM) was partly effective at 3 μM. The combined administration of FK 888 (10 μM) and GR 94,800 (3 μM) or GR 82,334 and GR 94,800 abolished the nonadrenergic noncholinergic contraction. The response to a prolonged period of stimulation (3 Hz for 5 min) was evenly depressed by FK 888 or GR 82,334, while GR 94,800 was more effective in inhibiting the late (87% inhibition) than the peak response (25% inhibition). In the presence of nifedipine (1 μM) a marked inhibition of the nonadrenergic noncholinergic contraction was observed and a time lag was evident between stimulus application and onset of contraction, which showed slow onset and offset kinetics. The nifedipine-resistant nonadrenergic noncholinergic contraction was unaffected by FK 888 or GR 82,334 but was suppressed by GR 94,800. Submaximally effective (1–3 nM) concentrations of substance P and neurokinin A produced distinct patterns of contraction: the response to substance P was fast and declined rapidly toward baseline; the response to neurokinin A was slow and sustained. In the presence of nifedipine, the response to substance P was greatly depressed and became slower in onset; nifedipine did not affect the contraction to neurokinin A but slowed its time-course. In sucrose gap experiments, either a short (10 Hz for 1 s) or a prolonged period of electrical stimulation (3 Hz for 3 min) evoked membrane depolarization, action potentials and contraction: in response to the “prolonged” stimulation, distinct phasic and tonic component of contraction were observed. Nifedipine abolished action potentials and the phasic contraction produced by a short period of stimulation, reduced by about 50% the maximal contraction developed during the prolonged stimulation without affecting the amplitude of the tonic response. In the presence of nifedipine, GR 82,334 (3 μM) blocked the membrane depolarization but did not affect contraction; GR 94,800 (0.1 μM) did not affect deporalization but abolished contraction. The present findings demonstrate that both NK 1 and NK 2 receptors mediate the nonadrenergic noncholinergic excitation of the circular muscle of the guinea-pig colon to nerve stimulation and indicate a remarkable specialization of the two receptors in producing a “fast” neurotransmission, NK 1 receptor-mediated, which relies upon the activation of voltage-sensitive calcium channels, versus a “slow” type of neurotransmission, NK 2 receptor-mediated, which is largely independent from activation of voltage-sensitive calcium channels.

Electrical activity and contraction in cells isolated from rat and guinea‐pig ventricular muscle: a comparative study.

1. Contraction in single ventricular muscle cells from rat and guinea-pig heart was measured using an optical technique, while at the same time either action potentials were recorded or transmembrane currents were measured under voltage-clamp conditions. 2. When the membrane was depolarized to 0 mV, there was a phasic and a tonic component of the contraction in guinea-pig cells, whereas in rat cells only the phasic component was obvious. In both species the depolarizations evoked the second inward current (Isi). 3. In rat cells, when the membrane potential during a depolarization was varied over the range -40 to +60 mV, the amplitude of contraction first increased to a peak at a potential close to 0 mV, and then declined as the membrane potential became more positive. In contrast, contraction in guinea-pig cells measured under similar conditions continued to increase as the depolarization was increased, and the tonic component of contraction became more obvious at more positive potentials. Contraction amplitude in guinea-pig cells could also be increased by increasing pulse duration under conditions where the tonic component of contraction was prominent. 4. Contraction during depolarization was suppressed by ryanodine in rat cells, whereas in guinea-pig cells contraction persisted, but with a modified time course. Ryanodine did inhibit spontaneous contractions of guinea-pig cells during exposure to low extracellular sodium. 5. Nifedipine suppressed Isi and phasic contraction in both rat and guinea-pig cells. In guinea-pig cells these effects developed contemporaneously, but in rat cells substantial reduction of Isi occurred before marked suppression of contraction. 6. In rat cells exposed to strontium in place of external calcium, inactivation of Isi was slowed and contraction was prolonged, with a slower time-to-peak and relaxation. The time course of the action potential was modified and ryanodine no longer inhibited contraction of rat cells in the presence of strontium. 7. It is concluded that the amplitude of contraction in rat and guinea-pig ventricular cells is determined by calcium both entering through the surface membrane and released from internal stores, and that under normal conditions the balance is towards release from stores in rat cells, and towards entry through the surface in guinea-pig cells.

Norepinephrine-induced contractions of the rat aorta in the absence of extracellular calcium-III. Effects of cyclic nucleotides

1. 1. The rat aorta responds biphasically to norepinephrine (NE) in calcium-free medium. The dissociable phasic and tonic components of the contraction are mediated through α-adrenoreceptor activation and mobilization of intracellular calcium. 2. 2. Dibutyryl-cAMP, papaverine (which inhibits cAMP phosphodiesterase and increases cAMP levels), bromo-cGMP, and sodium nitroprusside (which stimulates guanylate cyclase and increases cGMP levels) inhibited in a concentration-dependent manner the biphasic aortic contractions induced by NE in calcium-free medium. 3. 3. The log IC 50 values of each of these smooth muscle relaxants for inhibition of the biphasic response to NE in calcium-free medium were very close to those required for inhibition of the contractions induced by NE or KCl in presence of extracellular calcium. 4. 4. The present findings indicate that the phasic and tonic components of NE-induced aortic contractions in calcium-free medium are subject to similar intracellular regulatory mechanisms by cyclic nucleotides at a step subsequent to α-adrenoreceptor occupancy by NE, since dibutyryl-cAMP and bromo-cGMP inhibited the tonic component of NE-induced contraction noncompetitively.

A comparison of contractile events induced by leukotriene D4 and histamine in smooth muscle—A function of calcium utilization

OF CONTRACTILE EVENTS INDUCED BY LEUKOTRIENE D 4 AND HISTAMINE IN SMOOTH MUSCLE-A FUNCTION OF CALCIUM UTILIZATION. S. R. Findlay, C.W. Parker, E.I. Bloomquist~ and C.R. Scheid~ Tufts University Sch06l of'Medicine, Boston, MA. Washington University School of Medicine, St. Louis, MO. and the University of Massachusetts Medical School, Worcester, MA. Studies characterize the time course of exogenous LTD4-induced smooth muscle contraction in guinea pig i leal longitudinal smooth muscle. Tissues were prepared and secured in a 37oc constantly aerated (95% 02-5% CO2) tissue bath. Parameters characterizing the speed of contraction were: latency time-from the agonist addition to force development; T 89 half the time between the end of L and maximum force; relaxation time (Tr) ended when tissues reached baseline passive tension (0.5 gm) after washing. LTD4 as compared to histamine had a longer latency time, a markedly longer T 89 Tm and Tr. For LTD4 (1.4 x lO-9M) Jn sec. + S.D. L=6.3+I.6, T 89 4.1, Tm= 84+47, Tr= 136u and forhistamTne L = 4.5+I.8, T 89 1.8+0.8, Tm 4.2+0.9, Tr= 12+6. Compared to histamine, LTD 4 appeared to activate only a late tonic component of contraction. Histamine induced an early phasic and late tonic component of contraction. The ID50 of D600 (a calcium channel antagonists) for histamine and LTD 4 contractile components supported the concept that the slowness of the onset of contraction is largely due to the inabi l i ty of LTD 4 to mobilize Ca z+ via the mechanisms used by histamine in the production of the phasic component of contraction. METABOLISM OF HUMAN C3 BY HUMAN MAST CELL TRYPTASE. L.B. Schwartz, M.D., Ph.D., J.J. Schratz, D. Vik, D.T. Fearon, M.D. and K.F. Austen, M.D., Boston, Massachusetts Human mast cell tryptase was assessed for peptidase act iv i ty against human C3 and the synthetic C3 analog, leu-ala-arg-p-nitroanalide (LAA) (Kabi) and esterase act iv i ty against tosyl-L-arg methylester (TAMe) and CBZ-lys-thiobenzyl ester (CLTE). Tryptase was purified to homogeneity after extraction from dispersed and concentrated pulmonary mast cells of 10-35% purity by chromatography on Dowex I-x2, DEAE-Sephadex and heparin-agarose. One unit (U) of act iv i ty catalyzes the cleavage of 1 ~mole of substrate/min at 22~ Human C3 was purified as in Biochemistry 15:4513, 1976. Michaelis Menton kinetics were observed with all synthetic substrates. Ester substrates at pH 8.1 had respective Km and Vmax values for TAMe of 0.35 mM and 124 U/mg and for CLTE of 0.024 mM and 118 U/mg. LAA at pH 7.4 had a Km of 2 mM and Vmax of 104 U/mg. Cleavage of C3 was assessed by incubation of 2 ~g of tryptase and 126 ug of C3 in 0.42 ml of 0.01 M Tris, pH 7.8, 0.15 M NaCI, 2 mM CaCl 2 at 37~ aliquots removed at time points up to 12 h were analyzed by SDS-PAGE under reducing conditions. The 70,000 MW 8 chain was not cleaved. Several cleavage products of the 110,000 MW ~ chain were detected at 26,000-38,000 MW and a single product was observed at about 9000 MW. The lower MW product had the same apparent MW as trypsingenerated C3a. Thus, tryptase from human mast cell secretory granules can metabolize human C3 and generate protein fragments which may amplify mast cell init iated reactions by a histamineindependent mechanism.

Effects of histamine on electrical and mechanical activity in voltage-clamped frog atrial fibres

The influence of histamine on electrical and mechanical activity of bullfrog atrial fibres was studied using a double sucrose gap arrangement. At a concentration of 2 × 10 −7 m histamine, duration and amplitude of the membrane action potential were decreased, upstroke velocity was increased. Voltage clamp measurements showed related alterations of the membrane currents: peak sodium inward current and late outward current were increased. Slow inward (Ca) current and phasic tension associated with short-lasting depolarizations were reduced. In contrast, tonic contraction during sustained depolarization was increased. Flux measurements with radioactive isotopes revealed a histamine-induced rise of [Na] i and a fall of [K] i. The positive inotropic effect of histamine on the tonic component of contraction seems to be secondary to the enhancement of [Na] i.