Lymphatic Smooth Muscle Research Articles

The effect of known K+-channel blockers on the electrical activity of bovine lymphatic smooth muscle

The effects of known K+-channel blockers on the electrical properties of bovine lymphatic smooth muscle were investigated using the double sucrose-gap technique. Constant current anodal pulses elicited hyperpolarizing electrotonic potentials (EP's) which were characterised by a "sag" in the potential record. Current/voltage relationship (I/V), which were examined by measuring EP amplitude at the end of 5 s anodal pulses (less than 30 microA), showed an apparent increase in conductance with increasing hyperpolarization. In the presence of caesium (10 mM), 4-aminopyridine (10 mM) or in the absence of external K+ the sag in the EP was lost and the inward rectification characteristic of the control I/V relationship was abolished. Barium (2.5 mM) also abolished in sag in the EP although TEA (10 mM) had no effect on either EP shape or I/V relationship. Thus it would appear that lymphatic smooth muscle shows inward rectification which is slowly activating and is blocked by some of the known K+-channel blockers or by the removal of external K+.

Mechanism of alpha-adrenergic excitation in bovine lymphatic smooth muscle.

Measurements were made, using the double sucrose-gap technique, of electrical and mechanical responses of bovine lymphatic smooth muscle to constant current pulses. After beta-blockade with 10(-6) M propranolol, stimulation of the alpha-receptors with norepinephrine (5 X 10(-6) M) depolarized the membrane and decreased membrane conductance. The depolarization and decrease in membrane conductance persisted in Li+- and choline-substituted low-Na+ solution, in methanesulfonate-substituted low-Cl- solution, and in Ca2+- free solution containing 1 mM ethyleneglycol-bis(beta-aminoethylether)-N, N'-tetraacetic acid. Tetraethylammonium (10 mM) did not itself affect membrane resistance nor did it block the increase in resistance due to norepinephrine. In contrast, cesium (10 mM) increased membrane resistance and prevented norepinephrine from increasing this further. As well as these effects on membrane resistance, norepinephrine (5 X 10(-6) M) increased the duration of the action potential, and this was accompanied by an increased force of contraction. Tetraethylammonium prolonged the action-potential plateau and potentiated norepinephrine's effect. These results suggest that norepinephrine is likely to increase the efficiency of lymphatic pumping due to both its positive inotropic effect and the improved safety margin for propagation resulting from the increase in membrane resistance. The latter effect may be due to the suppression of an outward K+ current.

Beta-adrenergic inhibition of bovine mesenteric lymphatics.

The beta-action of catecholamines on lymphatic smooth muscle was studied by observing the effect of isoprenaline on electrical and mechanical activity in the double sucrose-gap. Action potentials and phasic contractions evoked by depolarizing pulses were abolished within 2 min of drug addition. Isoprenaline hyperpolarized the membrane and increased membrane conductance. Tetraethylammonium (10 mM) did not itself affect membrane resistance but reduced the hyperpolarization and the increase in conductance caused by isoprenaline. Removal of K+ from the external solution reduced membrane conductance and increased the hyperpolarization due to isoprenaline. When the NaCl content of Krebs solution was replaced with LiCl or choline chloride, isoprenaline no longer blocked action potential firing and its effects on phasic contractions and membrane conductance were reduced. In contrast, ouabain (10(-5) M) did not block the effect of isoprenaline on membrane potential and membrane conductance. These results suggest that beta-adrenergic inhibition of lymphatic smooth muscle involves an increase in an outward K+ current, though an additional metabolic effect cannot be ruled out.

Pre- and postjunctional α-adrenoceptors at sympathetic neuroeffector junction in bovine mesenteric lymphatics

We studied isolated bovine mesenteric lymphatics to elucidate the pharmacological characteristics of pre- and postjunctional α-adrenoceptors at the sympathetic neuroeffector junction. Cylindrical strips were incubated with [ 3H]-noradrenaline and mounted for superfusion. Electrical stimulation (2 Hz, 0.5 msec, 50 V) augmented the fractional release of labeled noradrenaline. Exogenous noradrenaline and clonidine caused a depression of the evoked tracer release. Phenoxybenzamine and yohimbine markedly enhanced the evoked overflow of adrenergic transmitter. Rings of lympahatic vessels were mounted for isometric tension recording in organ chambers filled with Krebs-Ringer bicarbonate solution. The vessels contracted when exposed to phenylephrine and clonidine. The ED 50 of clonidine was significantly lower than that of phenylephrine. Prazosin caused a parallel shift to the right of the dose-response curve to phenylephrine. The antagonist, however, suppressed the magnitude of the maximum response to clonidine. Yohimbine caused parallel shift to the right of the dose-response curves to phenylephrine and clonidine, respectively. The Schild plots for yohimbine demonstrated that the drug was a competitive antagonist to phenylephrine and clonidine. The pA 2 value of yohimbine to clonidine (7.6 ± 0.4) was larger than that to phenylephrine (6.2 ± 0.4). The pA 2 value of prazosin to phenylephrine was 7.2 ± 0.3. These results suggest that prejunctional α-adrenoceptors are involved in the negative feedback mechanism for autoregulation of noradrenaline release during post-ganglionic sympathetic nerve stimulation, and that both α 1- and α 2-like adrenoceptors do exist on lymphatic smooth muscle cells.

Variegated effects of prostaglandins on spontaneous activity in bovine mesenteric lymphatics

The effects of PGs (PGA 2, PGB 2, PGE 2, PGF 2α, PGI 2, and 6-keto PGF 1α) were investigated quantitatively in insolated bovine mesenteric lymphatics for elucidating the possible significance of PGs in lymph propulsion. 10 −8 M PGF 2α, PGA 2, and PGB 2 produced recognizable increases of frequency and amplitude of spontaneous contractions. The positive chrono-and inotropic responses were enhanced with increasing concentration of the PGs from 10 −8 to 10 −6 M. Both PGE 2 and PGI 2 in a concentration range from 10 −8 to 10 −6 M caused dose-related reductions in the amplitude of spontaneous contractions. No decrease in contraction rhythm was observed by the administration of PGI 2 and PGE 2 at concentrations lower than 5 × 10 −7 M. All of the above-mentioned responses induced by PGF 2α, PGA 2, PGB 2, PGI 2, and PGE 2 were not inhibited by pretreatment with α- and β-adrenergic blocking agents, muscarinic antagonist, antihistamics, and serotonic antagonist. These findings suggest that PGF 2α, PGA 2, and PGB 2 in a low concentration may facilitate lymph flow of bovine mesenteric lymphatics in the living body, since the spontaneous contractions of lymphatic smooth muscle may drive lymph centripetally in the presence of direction valves. On the other hand, PGI 2 and PGE 2 may cause an inhibition of lymph transport.

Effect of potassium on adrenergic nerve endings in bovine mesenteric lymphatics

This work concerned the effect of KCl (60 mM) on noradrenaline efflux from aminergic nerve endings in bovine mesenteric lymphatics. In high potassium Krebs solution containing cocaine (10 −7M), the lymphatics showed some phasic contractions followed by a tonic contraction with a high initial peak. The high KCl solution also caused a transient but statistically significant increase of tritiated noradrenaline efflux ( P < 0.01) which was not blocked by tetrodotoxin (10 −7 M). However, 5 × 10 −6 M 5-hydroxytryptamine or 10 −5 M prostaglandin F 2α produced a contraction of the lymphatic smooth muscle similar in magnitude to the contraction by 60 mM KCl. In this case, however, no increase was observed in 3H efflux. A Caantagonist, D-600 (10 −4 M) inhibited the potassium-induced rise in tritiated noradrenaline efflux and suppressed dose-dependently the contractile response to high potassium solution. These observations support the suggestion that the effect of KCl on 3H-release reflects an effect of kinetics of exchange across the nerve ending rather than an effect of contraction per se on isotope exchange through the complex extracellular matrix, and that the activation of Ca channels in the adrenergic nerve terminals may contribute to the release of noradrenaline in high KCl solution.

Effects of arachidonic acid and its cyclo-oxygenase and lipoxygenase products on lymphatic vessel contracility in vitro

Lymphatic vessels exhibit rhythmical contractility in vivo and in vitro and this activity appears ti regulate lymph flow. A technique for measuring the cicurlar muscle contractions of isolated bovine mesenteric lymphatic vessel segments has been devised and utilized to study the pharmacological properties of these vessels. Non-contracting lympahtic vessels can be induced to contract rhythmically with a variety of mediators, the most potent being a stable PGH 2 analogue (compound U46619), and the leukotrienes B 4, C 4 and D 4 (threshold concentrations in the nanomolar range). Prostagladin F 2α, noradrenaline, serotonin and histamine also elicited rhythmical activity but much higher concentrations were required. PGE 2 and PGE 1 were potent inhibitors of spontaneous contractions or those induced with U46619. In keeping with the diverse pharmacological effects of the metabolites of arachidonic acid, the addition of arachidonate to an isolated lymphatic vessel generated both stimulatory and inhibitory activities. It is concluded that arachidonic acid products (produced in the lymphatic vessel or entering the vessel in lymph draining the tissues) regulate lymph flow through their effects on lymphatic smooth muscle.

Effect of potassium on membrane potential and tension development in bovine mesenteric lymphatics

The transmembrane potential and tension of the smooth muscle of isolated bovine mesenteric lymphatics were recorded in solutions having various potassium concentrations. The relation between the membrane potential and the logarithm of the external K + concentration was observed. The maximum slope was 39 mV per 10-fold change in [K +] 0. The minimum depolarization necessary for inducing contraction was 6 mV. The maximal contraction was recorded at a [K +] 0 of 60 m M. In K-free solution, the membrane depolarized by about 9 mV, and the muscle developed a sustained tension. The addition of 10 −5 M ouabain to normal Krebs solution caused a depolarization of the membrane and a sustained contraction. The depolarization and tension development in the K-free solution were potentiated by 10 −5 M ouabain. These observations suggest that changes of membrane potential may play a major role in the activation of the contractile protein in lymphatic smooth muscle and also that there is an electrogenic sodium pump in the membrane. The depolarization and tension development in K-free solution may be due to a decreased activity of the electrogenic Na pump.

Active and passive mechanical characteristics of bovine mesenteric lymphatics.

Pressure-volume and pressure-radius relationships in lymphangions isolated from bovine mesenteric lymphatics were similar in pattern with those in the vein. Circumferential modulus of elasticity of the lymphatics ranged from 4.2 x 10(4) tatic walls. The contractile force increased in early stages of distension and decreased after an optimal intraluminal pressure was attained. The spontaneous activity was also affected by the rate of wall deformation. The pacemaker site of spontaneous activity seemed to be in the wall in the immediate vicinity of the inlet valve of a lymphangion. The activity propagated with a velocity of 4-5 mm/s. Ejection fraction of a lymphangion was between 45 and 65%. The endurance limit of the lymphatic valve was 68.4 +/- 7.6 cmH2O in specimens of about 3 mm in outer diameter. These findings suggested that lymphatic smooth muscle seemed to play a major role in elastic behavior of the wall and in regulation of the spontaneous activity, thereby affecting significantly passive and active lymph transport.

Electrical field stimulation as a method of stimulating nerve or smooth muscle in isolated bovine mesenteric lymphatics.

The purpose of this study was to determine the parameters of transmural stimulation which could be used to selectively stimulate intramural nerves or lymphatic smooth muscles of isolated bovine mesenteric lymphatics with a view to studying the innervation of these vessels. Isometric contractions were elicited in quiescent vessels either by 10s trains of pulses at various nominal voltages (10–100 V), frequencies (0.5–96 Hz) and pulse width (0.1–10 ms), or by a single pulse of 1 s duration at 50 V. The chronaxie of strength-duration curve at 1 Hz was 156±27 μs (n=6). The frequency-response curve at 50 V and 0.3 ms in pulse width showed that threshold response was obtained at 1 Hz and was maximal at 24 Hz. In the presence of 10−7 M tetrodotoxin or 10−6 M phenoxybenzamine and 10−6 M propranolol, the curve was moved to the right and down, and the threshold and maximal responses were obtained at 6 and 96 Hz, respectively. The pulse width-response relationship at 2 Hz and 50 V demonstrated that two kinds of S-shaped curve. The curve changed to a single S-shape curve with these drugs, which had a threshold pulse width of 0.5 ms. The responses to the single pulse of 1 s duration were not affected by high concentrations of tetrodotoxin or phenoxybenzamine. The responses were abolished with Ca-antagonist, 10−4 M D-600 These results suggest that it is possible to selectively activate intramural nerves or lymphatic smooth muscles by choosing the appropriate parameters of electrical stimulation. Thus, the optimal parameters of electrical stimulation of intramural nerves of the lymphatics are at a frequency of 2 Hz, with a voltage from 20–50 V, and a pulse width between 0.1 and 0.4 ms. The characteristics of innervation in bovine mesenteric lymphatics are also discussed.

The response of lymphatic smooth muscles to vasoactive substances.

A study was made of the isotonic response of bovine mesenteric lymphatics to several physiological vasoactive substances. Contractions of lymphatic smooth muscles were induced by serotonin (5-HT), prostaglandin F2alpha (PGF2alpha), noradrenaline (NA), histamine, dopamine and acetylcholine (ACh). The smooth muscles were particularly sensitive to 5-HT. Excepting PGF2alpha no other substances could equal 5-HT in the magnitude of the maximum response. The majority of 5-HT receptors seemed to be the D receptors. The decreasing order of the contractile responses was as follows: 5-HT greater than PGF2alpha greater than NA greater than histamine greater than dopamine greater than ACh. The contractile response to ACh was observed only in specimens involving valvular region. It was very likely that, in the lymphatics, there were 2 kinds of receptors for catecholamines, i.e. alpha and beta receptors, and the stimulation of the former induced smooth muscle contraction and that of the latter relaxation. A difference was noticed between the responses of valvular and intervalvular segments to NA. Relaxations of lymphatic smooth muscles were induced not only by isoproterenol but also by adenosine and adenine nucleotides. The decreasing order of the relaxant responses was as follows: ISP greater than adenosine greater than ATP greater than ADP greater cyclic AMP greater than or equal to AMP. The relaxant responses to adenine nucleotides tended to reduce with decrease in the number of high energy phosphates.

Contractile stimuli in collecting lymph vessels.

Contractility was investigated in collecting lymph vessels of rat and guinea pig mesentery. Lymphatic diameter and micropressure were simultaneously recorded under normal conditions and during micromanipulation of intralymphatic pressure. Spontaneous activity involved characteristic changes in intralymphatic pressure coincident with opening and closure of upstream and downstream valves. Contractions were irregular but still predictable by noting the trend of intraluminal pressure as it approached a threshold level. Mechanical obstruction upstream of lymph vessels reduced lymph pressure and contraction frequency. Microinjection or withdrawal of fluid caused contraction frequency. Microinjection or withdrawal of fluid caused contraction frequency to rise and fall, respectively. Contraction rate was not affected by the level of general anesthesia. Lymphatic wall tension, as calculated from pressure-radius variables, correlated well with contraction frequency, suggesting a myogenic origin for the contractile mechanism. However, lymphatic smooth muscle may be inherently unstable since some contractility persists despite an absence of pressure stimuli.

Electrical activity of lymphatic smooth muscles.

SummaryMembrane action potentials of bovine mesenteric lymphatics were recorded simultaneously with isometric contractions by the use of the sucrose gap method. The action potentials, about 3 sec in duration, always had one-to-one correspondence to the contraction waves. Each action potential consisted of three phases, i.e., initial slow depolarization, spike, and slow repolarization. The application of nor-adrenalin elicited a slight but long-lasting depolarization superimposed by frequent discharges of action potentials. The administration of acetylcholine in a relatively high concentration to the preparations induced a transient depolarization followed by the occurrence of action potentials. From the effects of tetrodotoxin, manganese, calcium-free environment, and barium chloride on the spontaneous contractions, it was suggested that calcium current may probably play a major role in producing spike discharge in bovine lymphatic smooth muscles.

Vasa vasorum within the media of bovine mesenteric lymphatics.

SummarySmooth muscles in bovine mesenteric lymphatics were well-developed and arranged in three layers, i.e., the internal longitudinal, intermediate circumferential, and external longitudinal. The outermost one was much thicker than the other two. Blood capillaries were found within the smooth muscle layers as well as in the ad-ventitia. These blood capillaries may be essential for maintaining vigorous rhythmic contractions of the lymphatic smooth muscles which act as a driving force for the propulsion of lymph. The presence of numerous mitochondria and glycogen granules seemed to reflect a high metabolic activity of the smooth muscle cells.The authors are indebted to Dr. T. Suganuma and Mr. R. Ichikawa for their technical advice and assistance.