Muscle-specific enzyme activity patterns of the capillary bed of human oro-facial,masticatory and limb muscles

Enzyme-histochemical methods were used to analyse the activities of alkaline phosphatase (AP), dipeptidylpeptidase IV (DPP IV) and adenosine triphosphatase (ATPase) in capillaries of four different human oro-facial muscles, the major and minor zygomatic, the orbicularis oris and buccinator, one masticatory, the masseter and two limb muscles, the biceps brachii and first dorsal interosseus muscles. In all muscles, except for the orbicularis oris, the majority of the capillaries lacked enzyme activity. Therefore, none of these enzymes seems to be reliable as a general marker for human muscle capillaries. In general, the capillaries of the limb muscles and the major and minor zygomatic and the buccinator, were similar in their staining pattern for AP and ATPase, but differed in DPP IV staining. The orbicularis oris muscle differed from the other muscles by showing the largest proportion of capillaries with AP and ATPase activity. The masseter muscle had the largest proportion of capillaries stained for DPP IV. The muscle specific differences in enzyme activity of the capillaries are in agreement with our previous findings of specific differences between limb, oro-facial and masticatory muscles with respect to capillary supply and composition of fibre types and myosins. The results reflect functional specialization of the capillary bed of human muscles.     

Effects of strontium ions on contraction and action potential in rabbit papillary muscles. A comparison with effects of tetraethylammonium ions.

The effects of Sr2+ on contraction and action potential were studied in rabbit papillary muscles and compared with effects of tetraethylammonium (TEA+). The membrane potential was measured with KCl-filled microelectrodes and the contraction was simultaneously recorded using a mechanoelectrical transducer. A partial (90%) substitution of extracellular Ca2+ (Ca2+e) by Sr2+ produced stimulation frequency-dependent prolongation of the action potential (AP) with a dominant phase "plateau" as well as prolongation of the contraction. At low frequencies where the AP prolongation was well pronounced, the contraction became biphasic. The effect of Sr2+ on both AP and contraction was blocked by nifedipine (10 mumol/l) or by increasing Ca2+e. Ryanodine suppressed the early contraction component only. AP was prolonged to a similar extent and in the same frequency-dependent manner by TEA+ (20 mmol/l). Despite similar AP configuration, no biphasic contraction developed in the presence of TEA+. High Ca2+e (10 mmol/l) or low Na+e (70 mmol/l) suppressed the TEA+ effect on AP. The data indicate that the two components of the biphasic contraction are of different origin; the early one is activated by activator cation released from the sarcoplasmic reticulum while the late one results from the Sr2+ entry across the sarcolemma via L-type Ca2+ channels.     

Morphology and physiology of capillary systems in subregions of the subfornical organ and area postrema

From recent morphological and physiological studies of capillaries, I shall review four new or revised concepts about blood-tissue communication in the subfornical organ (SFO) and area postrema (AP). First, the capillary systems of SFO and AP exhibit subregional differentiation correlated topographically with cytoarchitecture, densities of immunoreactivity for several peptides and amines, cellular sensitivity to neuroactive substances, afferent neural terminations, and tissue metabolic activity. Thus, contrary to frequent citations, the angioarchitecture and microcirculatory physiology of these small sensory nuclei are not homogeneous. Second, electron microscopic, morphometric, and topographical studies reveal that SFO contains three different types of capillary and AP has two. The differentiated capillary morphology appears to be well organized for specialized functions particularly in SFO subregions. No other body organ or small tissue region is known to have such capillary diversity, further highlighting the complex functions served by SFO. Third, pools of interstitial fluid (Virchow-Robin spaces) surrounding type I and III capillaries in SFO and AP may participate in the receptive properties of these organs as low-resistance pathways for rapid dispersion of blood-borne hormones inside their organ boundaries. The parenchymal walls of Virchow-Robin spaces appear to harbour metabolic mechanisms for hormones such as angiotensin II, and thus could vastly extend the effective blood-brain surface area of permeable capillaries in SFO and AP. Fourth, SFO and AP bear similar physiological characteristics of high blood volume, yet relatively low rates of blood flow. Accordingly, intracapillary blood velocity must be quite slow in these organs, and the duration of transit by blood and circulating messengers rather protracted. This feature of slow blood transit time likely compounds the sensory capability of SFO and AP, rendering increased contact time for blood-borne hormones to penetrate the permeable capillaries of these structures and interact with their known dense populations of receptors for several homeostatic substances involved in regulation of blood pressure and body fluids.     

Enzymatic differentiation of arterial and venous segments of the capillary bed during the development of free muscle grafts in the rat

The revascularization of freely grafted muscles in the rat was studied by histochemical reactions that on frozen sections stain the arterial part of the capillary bed blue (alkaline phosphatase [AP] reaction) and the venous part of the capillary bed red (dipeptidyl peptidase IV [DPP IV] reaction). In 112 rats the extensor digitorum longus or soleus muscles were freely grafted and removed at various times up to 93 days following the surgery. In cross section, the capillaries of a normal muscle show a mosaic pattern of staining for the activity of the two enzymes. After grafting, DPP IV activity of capillaries is lost throughout the entire graft within a day or two; but within ischemic muscle, weak and diffuse AP staining persists in capillary remnants for up to 6 days. In the very periphery of the graft AP staining is also preserved in partially damaged capillaries. By 4 days, new AP-positive capillaries can be identified at the periphery of the graft, and in succeeding days AP-positive capillaries are found progressively nearer the center of the graft. At 7 days, the capillary/muscle-fiber ratios are 66% of normal in the periphery of the graft and 44% in the intermediate zone. DPP IV-stained capillaries are not seen until 7 days after grafting. By 60 days, when the grafts have become stabilized, the mosaic pattern of capillary staining has become reestablished. In mature grafts, the number of capillaries per unit area was slightly higher than that in control muscles, but the capillary/muscle-fiber ratio was slightly lower, owing to the smaller than normal cross-sectional areas of the regenerated muscle fibers.     

Enzymatic heterogeneity of the capillary bed of rat skeletal muscles

This study of the capillaries in rat skeletal muscle involved the use of a histochemical method that allows one to distinguish between arterial and venous portions of capillaries. Under controlled staining conditions, the arterial portion of the capillary bed reacts positively for alkaline phosphatase (AP) activity, and the venous portion is positive for dipeptidyl peptidase IV (DPP IV) activity. A short transitional capillary segment is positive for the activity of both enzymes. Capillaries of the normal soleus muscle and the red and white portions of the sternomastoid muscle have been quantitatively analyzed. Quantitative data demonstrated differences in capillary dimensions among the muscles studied. Capillaries of the white part of the sternomastoid were the longest, and they had the shortest DPP IV-positive segment (8% of the total capillary length). Capillaries of the soleus muscle were the shortest, and they also had short DPP IV-positive segments (16%). In contrast, the DPP IV-positive segments of the red part of the sternomastoid occupied 60% of the total capillary length. Survey cross sections reveal a mosaic distribution of patches of capillaries stained for AP and DPP IV activity. This study reveals that within given bundles of muscle fibers, the capillaries that run parallel to the muscle fibers are aligned relative to one another in such a manner that their arterial and venous segments are in register.     

Alkaline phosphatase and dipeptidylpeptidase IV staining of tissue components of skeletal muscle: a comparative study.

A combined alkaline phosphatase (AP) and dipeptidlypeptidase IV (DPP IV) staining reaction has demonstrated enzymatic heterogeneity of the arterial and venous segments of capillaries in rat skeletal muscle. This study compared the staining reactions of skeletal muscles in many commonly used laboratory animals, including the axolotl, chick, quail, Monodelphys, rat, mouse, hamster, guinea pig, rabbit, dog, monkey, and human. DPP IV activity was found in the venous ends of the capillaries and in the endothelium of some larger veins in many of the species but was never demonstrated in the arterial side of the circulation. AP was found in the arterial ends of capillaries in all species except the axolotl, and it was also found in the endothelium of larger arteries of most species. AP activity was absent in venous endothelium of all species except for birds and Monodelphys. DPP IV activity was found in the perineurium of intramuscular nerves of most species, and AP activity was commonly seen in tendons and intramuscular connective tissue. The interspecies variability found in this study shows that care must be taken in comparing experimental data involving this technique from one species to another, but within a species the technique allows a fine level of discrimination between functionally distinct compounds of skeletal muscle tissue.     

Comparative distribution of four lysosomal enzymes within various structures of the human placenta

The histochemical activity of 4 lysosomal enzymes, acid phosphatase (AP), nonspecific esterase (NE), aryl sulphatase (AS), and beta-glucuronidase (BG), was compared in following structures of the human placenta: syncytiotrophoblast, villous stromal cells, fetal capillaries and larger blood vessels, cells of basal plate, macrophages, and Hofbauer cells. In spite of a general similarity in distribution of the investigated enzymes, differences concerning particular structures were found. Thus positively stained granules in endothelia of capillary vessels were revealed only in the reaction for BG, although contours of capillaries were also outlined by the diffuse reaction product for AS. The muscular layer of larger vessels reacted strongly for AS and weakly for NE with the remaining reactions being negative. In syncytiotrophoblast, BG appeared much less active than the other 3 enzymes. The possible significance of the BG positive granules in endothelial of capillaries and of the occasional divergence in distribution of the classical lysosomal markers (AP and BG) is discussed.     

Effects of intermittent hypoxia on action potential and contraction in non-ischemic and ischemic rat papillary muscle

Although it has been reported that intermittent hypoxia had the anti-arrhythmia effect, little is known about the effects on the action potential (AP) and contraction of papillary muscle, as well as the mechanism of anti-arrhythmia. The purpose of present study is to observe the effects of intermittent hypoxia on action potential and contraction of papillary muscle in rat left ventricle simultaneously using conventional intracellular microelectrode and contraction recording. The effects of intermittent hypoxia on AP and contraction during ischemic solution perfusion were also investigated. After exposed to intermittent hypoxia (six hours daily) for 42 days (IH42), duration (APD20) of 20%, 50% (APD50) and 90% (APD90) repolarization of AP prolonged significantly compared with animals in control (Con). Effective refractory period (ERP) in IH42 also prolonged significantly. Perfused with mimic ischemic solution, the changes of electric and mechanical activities in IH42 and in 28 days exposure to intermittent hypoxia (IH28) were much smaller than that in Con and IH14. The result of the study suggested that intermittent hypoxia prolonged the APD and ERP, offered the resistance against the ischemic damage on myocardium, which may be the electrophysiological basis of the anti-arrhythmia of intermittent hypoxia.     

Remote arteriolar dilations in response to muscle contraction under capillaries

In hamster cremaster muscle, it has been shown previously that contraction of skeletal muscle fibers underlying small groups of capillaries (modules) induces dilations that are proportional to metabolic rate in the two arteriolar generations upstream of the stimulated capillaries (Berg BR, Cohen KD, and Sarelius IH. Am J Physiol Heart Circ Physiol 272: H2693-H2700, 1997). These remote dilations were hypothesized to be transmitted via gap junctions and not perivascular nerves. In the present study, halothane (0.07%) blocked dilation in the module inflow arteriole, and dilation in the second arteriolar generation upstream, the branch arteriole, was blocked by both 600 mosM sucrose and halothane but not tetrodotoxin (2 microM). Dilations in both arterioles were not blocked by the gap junction uncoupler 18-beta-glycyrrhetinic acid (40 microM), and 80 mM KCl did not block dilation of the module inflow arteriole. These data implicate a gap junctional-mediated pathway insensitive to 18-beta-glycyrrhetinic acid in dilating the two arterioles upstream of the capillary module during "remote" muscle contraction. Dilation in the branch arteriole, but not the module inflow arteriole, was attenuated by 100 microM N(omega)-nitro-L-arginine. Thus selective contraction of muscle fibers underneath capillaries results in dilations in the upstream arterioles that have characteristics consistent with a signal that is transmitted along the vessel wall through gap junctions, i.e., a conducted vasodilation. The observed insensitivities to 18-beta-glycyrrhetinic acid, to KCl, and to N(omega)-nitro-L-arginine suggest, however, that there are multiple signaling pathways by which remote dilations can be initiated in these microvessels.     

The fine structure of the area postrema of the sheep

The ultrastructural features of the area postrema (AP) were investigated in the suckling lamb, weaned lamb and adult sheep. No morphological differences were observed between lambs and sheep. Unciliated ependymal cells, linked by zonulae adherentes-type junctions and gap junctions, cover the AP ventricular surface. Clusters of pyriform neurons, glial cells, and axons are present in the parenchyma. The blood vessels are surrounded by wide perivascular spaces, which present an inner and outer basal lamina. The capillaries are of the fenestrated type. Perivascular glial cells rest on the outer basal lamina of the perivascular space and form a continuous ensheathment with their cell bodies or with flattened interdigitating processes. Along adjacent perivascular glial processes gap junctions are present. From our ultrastructural observations it appears that the overall cellular morphology of AP of the sheep does not differ substantially from that of monogastric mammals.     

Electromyographic basis of inaccurate movement; its dependence upon the mode of muscle contraction

The electromyographic basis of inaccurate performance was investigated in two rapid precision-grip skills controlled by concentric and eccentric muscle contractions respectively. Surface electromyograms, recorded from the first dorsal interosseous (DI), adductor pollicis (AP) and abductor pollicis brevis, were utilised to identify changes in the timing and intensity of muscle activation which may be responsible for inaccurate performance. The results showed that when fast precision-grip skills were controlled by concentric DI and AP muscle contraction, variations in the intensity of muscle contraction were responsible for inaccurate performance. However, when these skills were controlled by eccentric DI and AP muscle contractions, inaccurate performance resulted from variations in the timing of muscle activation. It was concluded that the nature of the deficiency in the patterns of muscle activation resulting in inaccurate performance was dependent upon the type of muscle contraction used in the skill.     

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.     

Some Characteristic Relaxant Effects of Aqueous Leaf Extract of Andrographis paniculata and Andrographolide on Guinea pig Tracheal Rings

The ethnomedicinal uses of the aqueous leaf extract of Andrographis paniculata Nees (AP) include treatment of pain and inflammation, malaria, asthma and common cold. We designed this study to characterize some effects of AP and those of its andrographolide constituent. Guinea pig tracheal rings suspended in organ baths containing PSS were precontracted with histamine or carbachol and then exposed to cumulative concentrations of AP, andographolide or theophylline. The effect of AP was tested in Ca2+-depleted tracheal rings stimulated with the EC50 of histamine in Ca2+-free PSS. IC50 and Emax values were calculated for each relaxant. Results showed that both AP and andrographolide possessed relaxant effects on the tracheal smooth muscle. While AP was more effective on histamine-induced contraction, andrographolide and theophylline were more effective on carbachol-induced contraction. The IC50 values of andrographolide were significantly higher than those of theophylline in the two contractile agents. The presence of AP significantly attenuated the contractile force produced by 6.4 x 10-3 M Ca2+ in Ca2+-depleted rings. It is concluded that andographolide contributes at least in part to the relaxant action of AP on tracheal smooth muscles. The mechanism of action is related to inhibition of Ca2+ influx into tracheal smooth muscle cells.     

Effect of ryodipine, fenigidin, verapamil, and manganese ions on the contractile strength and the myometrium potential duration during rhythmic stimulation and at rest

Action of the calcium channels blocking agents was studied using their strong but reversible concentrations, in different regimens. Riodypine and phenihydine suppressed the contraction strength and shortened the AP duration in the myometrium muscle stripes equally effectively both in resting and under stimulation (0.3 Hz). Verapamyl and manganese ions suppressed the contraction strength and shortened the AP duration more effectively under rhythmic stimulation than in resting.     

Activity of alkaline phosphatase in rat skeletal muscle localized along the sarcolemma and endothelial cell membranes

Alkaline phosphatase (AP), a membrane-associated glycoprotein which enhances the hydrolysis of monophosphate esters at alkaline pH, is widely distributed in animal tissues. AP activity is increased in a variety of muscle disorders, i.e., myopathies and denervation. Established histochemical methods at the light microscopy level failed to demonstrate AP in skeletal muscles. In the present study we applied the Gomori lead nitrate method for ultrastructural examination of AP in rat gastrocnemius muscles and showed that the enzyme was linked to the sarcolemma of the striated muscle and to the membranes of endothelial cells in adjacent capillaries. In comparison with ATPase activity, AP activity was inhibited by both levamisole and a pH of 7.2, but not by ouabain. Hence, it appears that in skeletal muscles AP is active at a high pH and is bound to cell membranes.     

Morphological changes in the mink area postrema during growth and under different stages of sexual activity

The histological features of the area postrema (AP) of mink brains of both sexes were investigated at different ages and physiological conditions with light and electron microscopy. The mink AP was a twin-winged structure located at the dorsal surface of the medulla oblongata and consisted of neurons, glial cells, and both continuous and fenestrated capillaries enmeshed in a rich neuropil. The ventricular surface of the mink AP was covered by a single layer of tanycytes except at its most caudal part that was covered by a basal membrane derived from the pia mater. Supraependymal cells and intraventricular axons were also a common finding over the apical poles of tanycytes. However, our study demonstrates that the mink AP acquires the above general features at an advanced postnatal time and that, once fully developed, it undergoes morphological changes that can be directly linked to the aging process and sexual activity of the animals.     

Electromechanical relationships of rabbit papillary muscle under interpolated extrasystole conditions and after a pause

The aim of the study was to describe and attempt to explain certain specific features of electromechanical coupling in the rabbit myocardium. Electromechanical correlations in the papillary muscles of the right ventricle of adult rabbit hearts were studied by a programmed stimulation technique. The duration of action potentials (AP) was measured in the plateau phase (Do, ms) and at -80 mV level (D80, ms), together with the intensity of the corresponding isometric contractions (MG, arbitrary units). After twenty AP of 1 Hz frequency, we interpolated an extrasystole with a variable interval (TE = 100-900 ms) and measured D0 and MG of the premature AP and the first AP of the subsequent cycle. When a steady state at 1 Hz frequency had been reached, we interpolated pauses (Tp) of 5 to 600 s and read D80 and MG of the first to the tenth contraction after the pause. The extrasystole D0 attained the maximum at TE = 260 ms and then fell abruptly. The MG of extrasystoles with a longer TE grew from the lowest value (0.3), attained 1 at TE = 700 ms and then remained stable. The first contraction after extrasystole displayed distinct postextrasystolic potentiation (MG = 2), while D0 was unwontedly short. Prolongation of TE was accompanied by an increase in its D0 value and by a steep drop in MG (to as little as 0.2). D80 of the first AP evoked after the pause fell proportionally to log Tp and then, from Tp = 60 s, gradually rose. The MG value of the first contraction after the pause fell proportionally to log Tp. The AP recovered much more rapidly than contractility from the effect of the pause. In the discussion, an attempt is made to explain the found correlations on the basis of differences in the behaviour of the calcium current channel system in the rabbit myocardium and a commentary on electromechanical correlations is based on the hypothesis that the free sarcoplasmic calcium level determines both membrane electrogenesis and the inotropic state.     

The action of ultrasound on the contraction strength and action potential of the papillary muscle of the rat heart

In experiments on isolated rat papillary muscles the effects of therapeutic doses of ultrasound (US) (intensity, less than 2 W/cm2) with frequency of 0.88 MHz on contraction force and action potential (AP) were studied. 12 muscles (from 14) responded to 3-min exposition of the US with a rise both in contraction force and in resting tension. Sensitivity to US and a value of inotropic effect changed significantly between the preparation, and the threshold intensities of US varied from 0.3 to 2 W/cm2. In 3 experiments the inotropic effect of US was more than 100%, but in others it was about 50%. Two preparations were not sensitive to the US. The positive inotropic effect of US was accompanied by membrane depolarization (up to 20 mV) and by prolongation of AP duration measured at 10% of its amplitude (APD10). The correlation between the increase in contraction force and APD10 was demonstrated. Some preparations responded to US with high depolarization (up to 50 mV) and were inexcitable. The US induced an increase in temperature less than 1 degree C, therefore all the effects of US could not be explained as a result of temperature rise.     

Influence of tongue muscle contraction and transmural pressure on nasopharyngeal geometry in the rat

The mammalian pharynx is a hollow muscular tube that participates in ingestion and respiration, and its size, shape, and stiffness can be altered by contraction of skeletal muscles that lie inside or outside of its walls. MRI was used to determine the interaction between pharyngeal pressure and selective stimulation of extrinsic tongue muscles on the shape of the rat nasopharynx. Pressure (-9, -6, -3, 3, 6, and 9 cmH?O) was applied randomly to the isolated pharyngeal airway of anesthetized rats that were positioned in a 4.7-T MRI scanner. The anterior-posterior (AP) and lateral diameters of the nasopharynx were measured in eight axial slices at each level of pressure, with and without bilateral hypoglossal nerve stimulation (0.1-ms pulse, 1/3 maximal force, 80 Hz). The rat nasopharynx is nearly circular, and positive pharyngeal pressure caused similar expansion of AP and lateral diameters; as a result, airway shape (ratio of lateral to AP diameter) remained constant. Negative pressure did not change AP or lateral diameter significantly, suggesting that a negative pressure reflex activated the tongue or other pharyngeal muscles. Stimulation of tongue protrudor muscles alone or coactivation of protrudor and retractor muscles caused greater AP than lateral expansion, making the nasopharynx slightly more elliptical, with the long axis in the AP direction. These effects tended to be more pronounced at negative pharyngeal pressures and greater in the caudal than rostral nasopharynx. These data show that stimulation of rodent tongue muscles can adjust pharyngeal shape, extending previous work showing that tongue muscle contraction alters pharyngeal compliance and volume, and provide physiological insight that can be applied to the treatment of obstructive sleep apnea.     

Computational biology in the study of cardiac ion channels and cell electrophysiology

The cardiac cell is a complex biological system where various processes interact to generate electrical excitation (the action potential, AP) and contraction. During AP generation, membrane ion channels interact nonlinearly with dynamically changing ionic concentrations and varying transmembrane voltage, and are subject to regulatory processes. In recent years, a large body of knowledge has accumulated on the molecular structure of cardiac ion channels, their function, and their modification by genetic mutations that are associated with cardiac arrhythmias and sudden death. However, ion channels are typically studied in isolation (in expression systems or isolated membrane patches), away from the physiological environment of the cell where they interact to generate the AP. A major challenge remains the integration of ion-channel properties into the functioning, complex and highly interactive cell system, with the objective to relate molecular-level processes and their modification by disease to whole-cell function and clinical phenotype. In this article we describe how computational biology can be used to achieve such integration. We explain how mathematical (Markov) models of ion-channel kinetics are incorporated into integrated models of cardiac cells to compute the AP. We provide examples of mathematical (computer) simulations of physiological and pathological phenomena, including AP adaptation to changes in heart rate, genetic mutations in SCN5A and HERG genes that are associated with fatal cardiac arrhythmias, and effects of the CaMKII regulatory pathway and beta-adrenergic cascade on the cell electrophysiological function.