Guinea-pig Portal Vein Research Articles

ATP causes postjunctional potentiation of noradrenergic contractions in the portal vein of guinea-pig and rat.

Superfusion of the portal vein of rat and guinea-pig with Krebs' solution maintained at 25 degrees C greatly inhibits spontaneous contractions of the preparations and allows contractile responses to ATP and noradrenaline to be measured accurately. Under these conditions, continuous superfusion with ATP (10(-5) M), a concentration which had no effect on either basal tension or spontaneous activity, caused a significant shift to the left of the concentration-response curve to exogenous noradrenaline in both tissues. The mechanism of this potentiation induced by ATP may differ in the two tissues since in the rat portal vein potentiation appeared to be rapidly reversed by superfusing with ATP-free solution, whereas in the guinea-pig portal vein a further concentration-response curve to noradrenaline, in the absence of ATP, was still significantly shifted to the left compared with the control curve. However, potentiation in the rat portal vein may have had a longer duration than is suggested by the results since control concentration-response curves to noradrenaline in this tissue showed a progressive shift to the right which, although not significant, is likely to have affected the apparent time course of potentiation. It is concluded that ATP can potentiate contractions to exogenous noradrenaline in the portal vein of rat and guinea-pig via an, as yet, unidentified postjunctional mechanism.

Chapter 13 Purines as cotransmitters in adrenergic and cholinergic neurones

Publisher Summary This chapter focuses on the evidence for adenosine triphosphate (ATP) as a cotransmitter in adrenergic and cholinergic neurons. Several studies in the past have indicated that on using tritium-labelled adenosine and noradrenaline (NA), ATP is released together with NA from sympathetic nerves supplying the rabbit aorta and portal vein. The coexistence of NA and ATP has also been demonstrated in rabbit ear artery, dog basilar artery, and rabbit pulmonary artery. ATP, as well as NA, release from guinea-pig portal vein has been shown to be abolished following sympathectomy. It has also been indicated that ATP is released as a cotransmitter with NA from sympathetic nerves supplying the vas deferens, taenia coli, nictitating membrane, a number of blood vessels, and frog heart. The proportion of ATP to NA varies considerably in sympathetic nerves supplying different organs and in different species, and it is also likely that various peptides coexist with ATP and NA in different sympathetic nerves, particularly neuropeptide Y, but also somatostatin, enkephalin, vasoactive intestinal polypeptide, and vasopressin-like peptide. The cotransmission of ATP and NA involves different postjunctional mechanisms: ATP produces contractions via an electromechanical coupling mechanism involving voltage-dependent Ca 2+ channels, while NA produces contractions via a spike-independent mechanism, involving receptor-operated Ca 2+ channels. ATP and NA act synergistically to potentiate each other's actions, and they help each other terminate neurotransmission by acting on prejunctional receptors to inhibit the release of transmitter.

Positive interaction between leukotriene C 4 and histamine and other mediators on vascular tissues

The interaction of leukotriene C 4 (LTC 4) with the contractile activity of histamine (H), serotonin (5HT) and norepinephrine (NE) has been investigated in isolated vascular preparations. Threshold concentration of LTC 4 (5 × 10 −9 M) significantly potentiated the vasoconstricting effect of these compounds on guinea-pig pulmonary artery (GPPA). This phenomenon was long-lasting for H since it was still present 40 min after LTC 4 had been washed. FPL-55712 (10 −5M) counteracted the increased H response on GPPA induced by LTC 4. Potentiation of H activity due to LTC 4 was also observed on guinea-pig thoracic aorta (GPTA) indicating that LTC 4-induced hyperreactivity is not a phenomenon restricted to the pulmonary vascular bed. In the experiments carried out in presence of indomethacin (3 × 10 −6M), LTC 4 still potentiated H-induced vasoconstriction on GPPA, however the time course of the phenomenon was significantly shorter than that observed in absence of the cyclooxygenase inhibitor. The contractile activity of H and NE on guinea-pig portal vein (GPPV) was not potentiated by LTC 4 These results demonstrate that LTC 4 induces hyperreactivity of the arterial vascular tissue to vasoactive compounds and suggest that cysteinyl-leukotrienes may have pathological significance in the hemodynamic changes occurring during anaphylactic reactions. Preliminary experiments carried out on human intralobar pulmonary artery strongly support this hypothesis.

Inhibition of tension development and actomyosin ATPase activity in barnacle muscle by the Ca2+-indicator dye antipyrylazo III

We have investigated the effects of the Ca2+-indicator dye antipyrylazo III on: (1) tension development in myofibrillar preparations from barnacle depressor muscle; and (2) actomyosin ATPase activity in myofibrils/native actomyosin isolated from barnacle muscle and in actomyosin hybrids prepared from pure (unregulated) rabbit F-actin and purified barnacle myosin. In all solutions, pCa was either heavily buffered with suitable [CaEGTA]/[EGTA] and/or measured with a calcium-electrode so as to offset the appreciable Ca2+-buffering effects of the dyes. Antipyrylazo III produced a rapid, reversible and concentration-dependent inhibition of: (1) tension development by isolated barnacle myofibrils; (2) calcium-regulated ATPase activity in barnacle myofibrils and native actomyosin; and (3) calcium-independent actin-activated ATPase activity in hybrid actomyosins prepared from purified barnacle myosin and rabbit actin. This latter observation indicated that the inhibitory effect of the dye on calcium-regulated tension and ATPase in intact myofibrils is due to specific repression of active crossbridge formation rather than modification of calcium-regulatory mechanisms. The hypothesis that antipyrylazo III specifically represses active crossbridge formation was supported by the observation that the dye had no effect on rigor tension development. Specific and saturable binding of the dye to these myofibrils was characterized by a maximum capacity of 1.3 mumol dye g-1 myofibrillar protein, consistent with a calculated 1 dye: 1 myosin stoichiometry. These various biological effects were observed with both commercially available antipyrylazo III and highly purified dye preparations. Preliminary studies using myofibrillar preparations from rabbit psoas muscle, guinea-pig portal vein smooth muscle, and scallop adductor muscle have indicated that contractile function in these muscle types does not appear to be inhibited by antipyrylazo III.

Release and recycling of calcium by the sarcoplasmic reticulum in guinea-pig portal vein smooth muscle.

The amplitude of interrupted contractions evoked by noradrenaline or caffeine in Ca2+-free, high-K+ solutions containing EGTA or La3+ was determined in small (40-60 micron thick) bundles of guinea-pig portal anterior mesenteric vein. Interrupted contractions were produced by removing the stimulating agent as soon as the amplitude of the tension record reached its peak. The distribution of intracellular Ca2+ was determined, with electron probe X-ray microanalysis, in cryosections of preparations frozen in the relaxed state and at the peak of noradrenaline-induced contractions. Interrupted contractions of maximal or near-maximal amplitudes could be evoked every 2 min for up to 15 min in the virtual absence of extracellular Ca2+. If noradrenaline was allowed to remain in the solution throughout the period of spontaneous relaxation, a subsequent contraction could no longer be evoked in the absence of extracellular Ca2+. Interrupted contractions, similar to those evoked by noradrenaline, could also be stimulated by caffeine. The amplitude of reproducible interrupted contractions in Ca2+-free, high-K+ solution was graded with noradrenaline concentration. The ability of these smooth muscles to contract repeatedly and maximally in Ca2+-free solutions indicates the recycling of Ca2+ released from an intracellular store. The occurrence of these contractions in high-K+ (depolarizing) solutions supports the conclusion (Devine, Somlyo & Somlyo, 1972) that the release of intracellular Ca2+ is one of the mechanisms of pharmacomechanical coupling. The number of subplasmalemmal regions in which high Ca concentrations (greater than 10 mmol/kg dry wt.) were detected, with approximately 75 nm diameter electron probes, was reduced in muscles frozen at the peak of contraction, from 4.7/cell periphery in the relaxed to 1.4/cell periphery in the contracted preparations. In freeze-substituted smooth muscles, in which the membranes of the junctional sarcoplasmic reticulum could be visualized, the regions containing high Ca were identified as part of the sarcoplasmic reticulum (s.r.), indicating that the s.r. is the store from which noradrenaline and caffeine release Ca2+.

Indirect evidence that purinergic modulation of perivascular adrenergic neurotransmission in the portal vein is a physiological process.

The effects of adenine nucleotides and nucleosides on the contractile response to perivascular nerve stimulation were compared in the isolated portal vein of rabbit, rat and guinea-pig. 2-Chloroadenosine was more potent than adenosine and ATP, which were equipotent in producing inhibition of neurogenic contractions in the rabbit and rat via prejunctional P1-purinoceptors. In contrast, neurogenic contractions of the guinea-pig portal vein were not inhibited by adenosine and were potentiated by 2-chloroadenosine and, to a lesser extent, by ATP. Fluorescence histochemical localization of quinacrine, which binds to high levels of ATP, revealed a dense perivascular nerve plexus in the portal vein of rabbit and rat but not of guinea-pig. After chemical sympathectomy, quinacrine-positive nerves persisted in the rabbit (supporting other evidence for the presence of purinergic nerves) but not in the rat (supporting other evidence for ATP as a cotransmitter in adrenergic nerves). It is concluded that a prejunctional purinergic modulatory mechanism operates in adrenergic neurotransmission in the portal vein of rabbit and rat but not guinea-pig, and it is suggested that this indicates a physiological mechanism.

Effects of noradrenaline and acetylcholine on electro-mechanical properties of the guinea-pig portal vein.

Effects of acetylcholine (ACh) and noradrenaline (NA) on mechanical properties of smooth muscle cells of the guinea-pig portal vein were investigated using intact and skinned muscle preparations. In some preparations, the electrical activity was also recorded. In addition to ACh and NA, the effects of caffeine, procaine, excess concentrations of [K]o and MnCl2 were investigated. NA enhanced the mechanical response due to increase in the spike generation, receptor activated depolarization and release of Ca stored in the cell. ACh also enhanced the mechanical response, but this agent had little effect on the Ca release from the storage sites. Caffeine and procaine (10 mM) depolarized the membrane and enhanced the electrical activity. Caffeine enhanced the mechanical activity due to an increase in the membrane activity and the release of Ca stored in the cell, while procaine inhibited the contraction. Procaine inhibited and caffeine accelerated the Ca-induced Ca-release mechanism in the cell. NA released the Ca from the storage sites to a greater extent than did caffeine or ACh. MnCl2 inhibited the spontaneous membrane activity and contraction; however, low concentrations of MnCl2 increased the caffeine- or NA-induced contraction in Ca-free solution. When the Ca-tension relationship was observed in saponin-treated skinned muscles, the minimum concentration of Ca required to produce the contraction was 10(-7)M, as observed in other vascular tissues. Caffeine released the stored Ca, Na and ACh had no effect on the Ca release and procaine inhibited the caffeine-induced Ca release in skinned muscles. It was concluded that the membrane properties of smooth muscle cells in the portal vein are much the same as observed in other spontaneously active visceral muscles. Differences observed in the actions of NA and ACh on mechanical properties seems to be mainly due to different receptor-operated Ca release mechanisms.

Actions of nitroglycerine on smooth muscles of the guinea-pig and rat portal veins.

1 Effects of nitroglycerine (NG) on the electrical and mechanical activities of smooth muscle cells of the rat and guinea-pig portal veins were studied by a microelectrode technique and an isometric tension recording method.2 The membrane potentials of smooth muscle cells in the rat and guinea-pig were -44.4 mV and -47.6 mV, respectively. In both species the smooth muscle cells generated spikes as burst discharges.3 In the guinea-pig portal vein, NG (2.8 x 10(-8) M) produced biphasic potential changes, an initial transient hyperpolarization followed by depolarization. The hyperpolarization suppressed and depolarization enhanced spike activities.4 From the changes in the membrane potential produced by NG in various concentrations of [K](o), [Na](o) or [Ca](o), it is postulated that the hyperpolarization is due to an increase in the K-permeability and the depolarization is due to an increase in the Na-permeability of the membrane.5 NG (2.8 x 10(-5) M) had no effect on the membrane activity of the rat portal vein.6 NG consistently suppressed mechanical activities generated in both tissues. The minimum concentration required to suppress the mechanical activity was lower in the guinea-pig than in the rat portal vein.7 NG suppressed the contraction due to noradrenaline more than that evoked by excess [K](o) in both species.8 From these experiments, it is concluded that NG relaxes the muscular bed of portal veins of both species. In the rat portal vein, suppression of mechanical activity had no causal relation to the membrane activity. In contrast, in the guinea-pig portal vein, suppression of mechanical activity was slightly modified by changes in the membrane activity, i.e. hyperpolarization additively contributes to the relaxation and depolarization slightly suppresses the relaxation.

COMPARATIVE PHARMACOLOGICAL AND HISTOCHEMICAL EVIDENCE FOR PURINERGIC INHIBITORY INNERVATION OF THE PORTAL VEIN OF THE RABBIT, BUT NOT GUINEA‐PIG

1 Intramural nerve stimulation elicited a powerful relaxation of the longitudinal muscle of the rabbit portal vein in the presence of atropine and guanethidine, but not of the guinea-pig portal vein.2 Intramural nerve stimulation of the rabbit portal vein produced a 13 fold increase in release of (3)H-adenyl compounds after preloading with [(3)H]-adenosine. About 50% of this release was abolished by guanethidine. All release was abolished by tetrodotoxin. No significant release of radioactive compounds was observed during intramural nerve stimulation of the guinea-pig portal vein in the presence of guanethidine, although there was a 6 fold increase in release of radioactivity in the absence of drugs.3 Histochemical studies using quinacrine, which binds ATP showed a fine fluorescent nerve plexus, nerve bundles, and ganglion cells in the rabbit portal vein, but not in the guinea-pig portal vein. This plexus was still present after chemical sympathectomy with 6-hydroxydopamine.4 Adenosine 5'-triphosphate (ATP) relaxed the rabbit portal vein, but usually produced a biphasic response, consisting of a contraction followed by a relaxation, of the guinea-pig portal vein.5 Prostaglandins E(1) and E(2) caused contraction of the rabbit portal vein. Indomethacin, a prostaglandin synthesis inhibitor, potentiated the relaxations of the rabbit portal vein produced by both non-adrenergic, non-cholinergic nerve stimulation and ATP.6 High concentrations of antazoline and phentolamine, which antagonize purinergic responses in the guinea-pig taenia coli, caused a loss of basal tone so that it was not possible to assess their effects on the responses of the portal vein to either non-adrenergic, non-cholinergic nerve stimulation, or ATP.7 Comparison of the results on the portal vein of the rabbit and guinea-pig provides support for the view that: (i) quinacrine fluorescence can be used to localize purinergic nerves and that the rabbit portal vein is supplied by these nerves; (ii) ATP is released from adrenergic nerve fibres, although, based on histochemical analysis, about 3 to 7 times less than is released from purinergic nerve fibres.

Neuroeffector maturity of portal veins from newborn rats, rabbits, cats and guinea pigs.

Isolated portal veins from newborn and adult rats, rabbits, cats and guinea pigs were studied isometrically. Spontaneous activity and induced responses to transmural nerve stimulation (NS), noradrenaline, tyramine, acetylcholine and BaCl2 were quantitated. In agreement with our previous observations the portal veins of the newborn rat was found to lack spontaneous activity and responsiveness to applied stimuli whereas the newborn rabbit's portal vein showed inconsistent and weak responses. In the neonatal cat and guinea pig portal veins the pattern of the spontaneous activity and induced responses resembled those found in vessels from adult animals. It is concluded that the portal vein maturity at birth in several respects dramatic neuroeffector development of the portal vein during the postnatal period in the rat corresponds to prenatal events in cats and guinea pigs which are more highly developed at birth. Consequently, the lack of responsiveness of the portal vein during the first week of life does not seem to be secondary to the hormonal and hemodynamic adjustments during the neonatal period. Rather, the postnatal development of the rat portal vein occurs according to a genetically predetermined schedule, possible in parallel with the ontogenesis of vascular control in general.

Effect of cold storage on the sensitivity of and calcium influx into rat, rabbit and guinea pig portal veins.

After cold storage treatment (5-7 days at 2 degrees C) the sensitivity to norepinephrine (NE) was increased in rabbit portal vein, decreased in rat portal vein and not altered in guinea pig portal vein. Cold storage decreased the sensitivity to methoxamine (ME), Ba++ and Ca++ in rat and guinea pig veins but not in rabbit veins and failed to alter the sensitivity to K+ in all veins tested. Cold storage increased 45Ca-influx while it reduced the NE content and 3H-NE uptake of all portal veins examined. NE and ME-induced 45Ca-influx was greater in cold-stored veins that in fresh veins from rabbit and guinea pig. K+ decreased 45Ca-influx in all cold-stored veins. These results indicate that cold storage induces a prejunctional supersensitivity in rabbit portal vein but not in guinea pig and rat portal veins. However, cold storage increased 45Ca-influx in all three veins.

Effects of Diltiazem on Guinea Pig Portal Vein in Hypertonic Solution

Effects of diltiazem were investigated on the isolated portal vein of the guinea pig in hypertonic solution. Hyperosmolarity was produced by sucrose. In contrast to a small and somewhat irregular spontaneous contraction in isotonic solution, the portal vein in hypertonic solution elicited a spontaneous contraction at regular intervals and with a large amplitude and low frequency. The spontaneous phasic contraction in hypertonic solution was not affected by tetrodotoxin, atropine and propranolol. Phentolamine also had no significant influence in some preparations. On the contrary, remarkable changes in the contractile activity were found when the extracellular concentration of CaCl2 was reduced from 2.5 to 1.3 or 0.6 mM. In calcium-free solution, the spontaneous contraction was completely abolished. In addition, the phasic contraction was suppressed by CoCl2. Thus, it is assumed that the spontaneous contractile activity of the guinea pig portal vein in hypertonic solution is myogenic in nature and Ca++ plays an essential role in the contraction. On the other hand, diltiazem suppressed the spontaneous phasic contraction in both isotonic and hypertonic solutions. The spontaneously generated and electrically evoked spikes in hypertonic solution were also inhibited by diltiazem. Furthermore, the effects of diltiazem on the mechanical activity and evoked spike were reversed by the addition of CaCl2. Therefore, it is inferred that diltiazem suppresses the spontaneous phasic contraction by inhibiting the mobilization of Ca++ which triggers the generation of the contraction.

Mechanisms by which smooth muscle sensitivity may be altered by calcium.

6-Hydroxydopamine (6-OHDA) increased the sensitivity of rat, rabbit and guinea pig portal veins to norepinephrine (NE), methoxamine (ME), barium (Ba++) and calcium (Ca++) but not to potassium (K+). Reserpine potentiated the responses of NE, ME and Ca++ but not Ba++ or K+ in rabbit and guinea pig veins and did not alter the sensitivity of rat veins to all agonists tested. Cocaine only potentiated the NE responses of the veins and decreased the sensitivity of rabbit and guinea pig veins to K+. 6-OHDA increased 48Ca-influx in all veins whereas reserpine increased 45Ca-influx only in rabbit and guinea pig veins. Cocaine failed to increase 45Ca-influx in all veins tested. NE, ME and K+ increased 45Ca-influx in the veins from the different animals. The agonist-induced 45Ca-influx was greater in most of the supersensitive veins than in the control veins. In veins that failed to develop supersensitivity, agonist-induced 45Ca-influx did not differ from that of the control veins. It is concluded that the development of super- and subsensitivity exhibits species variation and that the alteration in Ca++ influx would be consistent with the changes in sensitivity of the venous smooth muscle.

Comparison of the Excitatory and Inhibitory Effects of Angiotensin and Vasopressin on Mammalian Portal Vein

Angiotensin produced inhibitory and excitatory responses in guinea pig portal vein and excitatory responses in rabbit and rat portal vein. Vasopressin produced inhibitory responses in guinea pig and rabbit portal vein and excitatory and inhibitory responses in rat portal vein. In guinea pig portal vein, inhibition by angiotensin and vasopressin was associated with an increase in membrane potential which was, on average, smaller than that produced by isoprenaline, and with a suppression of spike discharges. Tachyphylaxis of the responses to angiotensin and vasopressin in portal vein was not observed and their effects were unaffected by tetrodotoxin, atropine and phentolamine plus propranolol.

Membrane potential and contraction of vascular smooth muscle (portal vein) during application of noradrenaline and high potassium, and selective inhibitory effects of iproveratril (verapamil)

Membrane potential, measured intracellularly, and tension development were recorded in preparations of guinea pig portal vein. Iproveratril (verapamil) (10 −5 mole/liter) suppresses all components of spontaneous electrical and mechanical activity and leads to a depolarization of about 15–20 mV. Of the noradrenaline (NA)-induced effects on membrane potential, iproveratril selectively blocks the spike component, leaving the depolarization unchanged. Spike discharges, stimulated by a K + depolarization (120 mmole/liter), are also blocked by iproveratril, while the K + depolarization itself remains unaltered. Tension development elicited by NA application is reduced to about 40% of the control by iproveratril, whereas K + -induced contractions are completely abolished. The iproveratril-resistant part of the NA contraction can also be elicited during K + depolarization, without significant changes of membrane potential. It may be concluded that the response to NA of portal vein smooth muscle consists of two different components.

The membrane properties of the smooth muscle of the guinea-pig portal vein in isotonic and hypertonic solutions.

The membrane properties of the longitudinal smooth muscle of the guinea-pig portal vein were investigated under various experimental conditions.1. In isotonic Krebs solution, the membrane potential (-48.7 mV), the maximum rates of rise and fall of the spike (4.6 and 2.3 V/sec respectively), the space constant (0.61 mm), the conduction velocity of excitation (0.97 cm/sec) and the time constant of the foot of the propagated spike (18.4 msec) were measured.2. The various parameters of the muscle membrane in the isotonic solution were compared with those in the hypertonic solution prepared by the addition of solid sucrose (twice the normal tonicity).3. When the muscles were perfused with hypertonic solution, marked depolarization of the membrane and increased membrane resistance occurred. These were probably due to reduction of the K permeability, increased internal resistance of the muscle and shrinkage of the muscle fibre.4. The membrane potential in isotonic and hypertonic solutions was analysed into two components, i.e. the metabolic (electrogenic Na-pump) and the ionic (electrical diffusion potential) component in the various environmental conditions.(a) In isotonic and hypertonic solutions, the membrane was depolarized by lowering the temperature or by removal of K ion from the solutions. When the tissues were rewarmed or on readdition of K ion, the membrane was markedly hyperpolarized. These hyperpolarizations of the membrane were suppressed by treatment with ouabain (10(-5) g/ml.), by warming to only 20 degrees C and by K-free solution.(b) The relationships between the membrane potential and the [K](o) in isotonic Krebs, in the hypertonic (sucrose) Krebs, in the Na-free (Tris) Krebs and in the Cl-deficient (C(6)H(5)SO(3)) Krebs were observed. The maximum slopes of the membrane depolarization against tenfold changes of [K](o) were much lower than that expected if it behaved like a K electrode.(c) In Na-free (Tris) solution, the membrane was not depolarized in isotonic condition but it was depolarized in hypertonic condition.5. The low membrane potential in hypertonic solution (-37 mV) compared with isotonic solution (-49 mV) was thought to be mainly due to suppression of K permeability of the membrane and not due to suppression of the metabolic component. The electrogenic Na-pump and the membrane potential of the portal vein was discussed in relation to other excitable cell membranes.

Membrane properties of the smooth-muscle fibres of the guinea-pig portal vein.

The membrane activities and the various characteristic constants of the smooth-muscle membrane of the guinea-pig portal vein were investigated with the micro-electrode technique.1. The mean membrane potential was -37 mV. Spontaneous discharges appeared as regular bursts of short trains of spikes alternating with silent periods, as a mixture of single spikes and bursts of spikes appearing continuously, or as regular spikes with low frequency.2. Spontaneous spikes with overshoot were frequently observed. The maximum rate of rise of the spike was 3.7 V/sec. The shapes of the spikes were classified into three different types, i.e. pace-maker type of spike, monophasic spike and spike with a hump during the falling phase.3. Tetrodotoxin (10(-5) g/ml.) did not influence the patterns of the spontaneous train discharges nor the shape of the spike.4. Extracellularly applied outward current elicited spikes which were either monophasic or had a hump on the falling phase. Inward current elicited break excitation of the spike.5. Current-voltage relations, produced by application of inward current pulses to the tissue and measured at various distances from the stimulating partition, were linear.6. The smooth-muscle membrane of portal vein showed cable-like properties. The mean space constant of the membrane was 0.52 mm; the mean time constant of the membrane calculated from the electrotonic potential was 330 msec.7. Conduction velocity of the spike measured by insertion of two micro-electrodes was 0.58 cm/sec.8. The time constant of the foot of the propagated spike was 27 msec. The time constant of the membrane calculated from the time constant of the foot of the spike and the conduction velocity was 310 msec.9. The membrane properties of longitudinal smooth muscle of the portal vein were discussed in comparison with other veins and various visceral smooth muscles.