Transmitter characteristics of cutaneous,renal and skeletal muscle small arteries in the rat

AIM: We studied transmitter characteristics of proximal and distal arteries supplying skin (saphenous artery and its medial tarsal branch), kidneys (terminal branches of renal artery and interlobar arteries) and skeletal muscle (proximal and distal sections of external sural artery). METHODS: Artery segments were mounted in an isometric myograph and intramural nerves were activated by electrical field stimulation. Adrenergic and purinergic components of the neurogenic response were blocked using phenoxybenzamine and alpha,beta-methylene adenosine triphosphate (mATP), respectively. RESULTS: Arteries from skin or kidney developed rapid and prominent neurogenic contractile responses, with half-maximal amplitude reached within 5-15 s; responses in proximal vessels were greater than in distal vessels. Arteries from skeletal muscle responded to sympathetic stimulation with a moderate contraction developing over 1 min or more, the response of distal segments was greater than that of proximal segments. In skeletal muscle vessels the sympathetically evoked contraction was completely blocked by phenoxybenzamine, whereas in skin and renal vessels it was the combined effect of noradrenaline and adenosine triphosphate (ATP). Given alone, mATP did not change the magnitude of the response to nerve stimulation, but increased its latency and also potentiated the response to exogenous noradrenaline. In all vascular beds, distal vessels were more sensitive to noradrenaline and mATP. CONCLUSION: It thus appears that the noradrenaline/ATP ratio of the sympathetic vasoconstrictor response differs between vascular beds in a way that is consistent with known differences in the selective regulation of regional vascular resistance by the sympathetic nervous system.     

ATP as a sympathetic co-transmitter in rat vasomotor nerves-further evidence that individual release sites respond to nerve impulses by intermittent release of single quanta

A combination of intra- and extracellular recording was used to study neurotransmitter release in sympathetic vasomotor fibres in rat femoral and mesenteric arteries. The results show that neurotransmission in these preparations is similar to that in the ‘short’ sympathetic nerves in guinea-pig vas deferens and the ‘long’ sympathetic nerves in rat tail artery in the following respects:

• 1 The intracellularly recorded excitatory junction potentials (EJPs) and the extracellularly recorded junction currents (EJCs) presumably are caused by ATP secreted as a sympathetic co-transmitter.
• 2 The stimulus-evoked and spontaneous EJCs share the same brief time-course, which is similar to that of spontaneous EJPs, but much briefer than that of stimulus-evoked EJPs.
• 3 ‘Successful’ nerve impulses appear to release single transmitter quanta.
• 4 The probability of activation of individual release sites is low (0.002–0.02).
• 5 The low release probability cannot be accounted for by failure of the nerve impulse to invade the terminals.

Moreover, it is also shown that application of tetrodotoxin to the medium within the recording electrode effectively abolishes transmitter secretion in the area enclosed by the tip of the electrode, indicating that the effective length constant for a passively propagating nerve action potential is probably very small and that activation of the release mechanisms in ‘long’ sympathetic nerve fibres seems to require that the varicosities are actively invaded.     

Transmitter characteristics of small mesenteric arteries from the rat

We have studied the neurogenic response of small mesenteric arteries from the rat to evaluate the involvement of possible co-transmitters under various modes of stimulation. Segments of small branches of the mesenteric artery were mounted in a myograph and the intramural nerves were activated with transmural electrical stimulation. A single stimulation of the nerves caused a contraction that was reduced by only 20% in the presence of adrenergic blocking agents (prazosin or phenoxybenzamine), whereas the steady-state response to continuous nerve stimulation of high frequency was reduced by 90-95%. In contrast, all responses to applied noradrenaline in doses up to at least 1 mM were eliminated by phenoxybenzamine treatment. The stable ATP analogue, alpha,beta-methylene ATP, reduced the response to a single nerve stimulation by 70%, but reduced the contraction caused by continuous high-frequency nerve stimulation by only 10%. None of these agents affected the response to applied neuropeptide Y (NPY). The response of relaxed vessels to nerve stimulation was totally blocked by the combination of an adrenoceptor-blocking agent and alpha,beta-methylene ATP, although even in this situation a further neurogenic response could be revealed in vessels precontracted with vasopressin. Responses to either single stimuli or brief burst stimulations were potentiated after high-frequency stimulation. Both the adrenergic and non-adrenergic components were enhanced to roughly the same extent. Also the potentiated response was eliminated by the combined application of prazosin and alpha,beta-methylene ATP. The non-adrenergic transmitter in the sympathetic nerves of small arteries thus appears to be the dominant transmitter during low-frequency nerve stimulation, causing rapid but phasic activation. Noradrenaline is the most important transmitter for higher frequencies, exerting slower but sustained contractions. The post-stimulatory potentiation affects both the adrenergic and the non-adrenergic part of the neurogenic response.     

Electrical field stimulation of rat mesenteric small arteries: force and free cytosolic calcium during neurogenic contractions and mechanisms of non-neurogenic relaxations

The effect of transmural electrical field stimulation (TEFS) of rat mesenteric small arteries was studied. Stimulation parameters were selected to cause tetrodotoxin (TTX) sensitive contractions. In arteries precontracted with PGF_2α in the presence of phentolamine, TTX insensitive relaxation could be induced by TEFS. The relaxing effect of TEFS required higher stimulation amplitude and duration than the contractions. Thus, by appropriately choosing stimulation parameters, contractile responses could be elicited which were little affected by any relaxing effect, while contractions were abolished by TTX at any stimulation conditions in the present study. The contractions were abolished by cold storage and almost completely inhibited by phentolamine. Thus, contractions were neurogenic and primarily caused by noradrenaline. At low frequencies, TEFS caused phentolamine sensitive increases in free cytosolic calcium with no contractions. At higher frequencies, there was a further increase in free cytosolic calcium, associated with contraction. Only at high frequencies, noradrenaline from nerves caused sensitization of the contractile filaments to free cytosolic calcium as during stimulation with exogenous noradrenaline. The relaxations were associated with decreases in free cytosolic calcium and were probably non-neurogenic since they were resistant to TTX, cold storage, capsaicin, and repeated stimulation. Furthermore, relaxations were almost completely abolished by increasing extracellular potassium to 40 mM or by adding tetraethylammonium chloride or 4-aminopyridine. Relaxations were also reduced by ouabain and potassium free conditions.     

Effect of endothelium-dependent stabilization of the pressure drop in small arteries

Laboratory of Biomechanics and Regulation of the Circulation, All-Union Cardiologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR, V. N. Smirnov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 107 No. 6, pp. 649–651, June, 1989.     

Noradrenaline-induced depolarization is smaller in isobaric compared to isometric preparations of rat mesenteric small arteries

The hypothesis was tested that wall tension can influence the membrane potential response to noradrenaline (NA) using isometric and isobaric vessel preparations of rat mesenteric small arteries. The resting membrane potential was significantly less negative in the isobaric (–49.7±0.5 mV, S.E.M., n=12 vessels) compared to the isometric preparation (–56.1±0.7 mV, n=10), although there was no difference in wall tension. The depolarization induced by 10^–5 M NA was 2.6-fold smaller in the isobaric preparation, where wall tension decreased, compared to the isometric preparation, where wall tension increased. Since wall tension decreases under isobaric conditions, but increases under isometric conditions, the latter finding can be explained by assuming that part of the NA-induced membrane potential change is wall tension dependent.     

Rhythmic contractions of isolated,pressurized small arteries from rat

The present study was undertaken to examine the influence of transmural pressure on vasomotion and to determine if any such influence was endothelium–dependent. Responses to changes in intravascular pressure of cannulated mesenteric small arteries were investigated under no–flow conditions. Both intact and endothelium–denuded arteries dilated passively when intravascular pressure was increased stepwise from 20 to 140 mmHg. When tone was induced by noradrenaline, pressure increase resulted only in dilatation, independent of endothelium. The sensitivity to noradrenaline was significantly increased in vessels without endothelium, indicating a relaxing influence of the endothelium. Rhythmic contractions in response to noradrenaline occurred in all intact arteries, but were absent when the endothelium was removed. The amplitude of the rhythmic contractions decreased significantly when transmural pressure was elevated. The frequency increased when pressure was elevated from 20 to 80 mmHg and then remained rather constant during further pressure increases. As shown previously in non–pressurized arteries, exogenous cyclic GMP induced oscillations in endothelium–denuded arteries. Pressure–related effects on vasomotion were not dependent on an intact endothelium. Ryanodine, ouabain or verapamil inhibited the rhythmic activity, confirming previous results in non–pressurized arteries. Thus, changes in transmural pressure can modulate vasomotion, but this effect does not appear to be mediated by the endothelium. Generation of vasomotion may depend on release of Ca²⁺ from intracellular stores, the activity of the Na⁺, K⁺–pump and transmembrane Ca²⁺ inflow in a pressurized artery as shown previously in these arteries under isometric conditions.     

Prejunctional β2-adrenoreceptor blockade reduces nerve stimulation evoked release of endogenous noradrenaline in skeletal muscle in situ

Prejunctional β-adrenoceptor-mediated modulation of endogenous noradrenaline (NA) overflow elicited by sympathetic nerve stimulation was studied in blood-perfused canine gracilis muscle in situ. An attempt was made to subclassify these β-adrenoceptors by comparing the effects of β₁-selective (metoprolol) and non-selective (propranolol) β-adrenoceptor blockade. Animals were pre-treated with desipramine and phenoxybenzamine in order to counteract possible influences of neuronal uptake and stimulation-evoked changes in vascular resistance on the diffusion of NA into the blood stream. Metoprolol did not decrease stimulation-evoked NA overflow, as compared with control experiments (?10 and ?8 %, respectively). However, propranolol reduced stimulation-evoked NA overflow by 30% in metoprolol pre-treated animals (P < 0.05 vs. control experiments). Both antagonists elevated basal perfusion pressure, suggesting that vascular post-junctional β₁-as well as β₂-adrenoceptors are present. Propranolol increased stimulation-evoked vasoconstriction in metoprolol pre-treated animals, indicating that neuronally released NA may activate postjunctional β₂-adrenoceptors under these experimental conditions. In conclusion, our findings suggest that NA release can be enhanced by activation of prejunctional β₂-adrenoceptors in vivo.     

Contribution of Na+/Ca2+ exchanger to the regulation of myogenic tone in isolated rat small arteries

The contribution of the Na⁺/Ca²⁺ exchanger to the myogenic vascular tone was examined in rat isolated skeletal muscle small arteries (A_SK) with pronounced myogenic tone and mesenteric small arteries (A_MS) with little myogenic tone. Myogenic tone was assessed by the vascular inner diameter at transmural pressures of 40 and 100 mmHg. To depress the Na⁺/Ca²⁺ exchanger, the extracellular Na⁺ concentration ([Na⁺]o) was lowered from 143 to 1.2 mM by substituting choline‐Cl for NaCl. The A_SK developed significant myogenic tone and constricted further in low [Na⁺]o. Nifedipine (1 μM ) reduced both myogenic tone and low [Na⁺]o‐induced contraction. Because the membrane potential of A_SK was not changed by low [Na⁺]o (–35 ± 2 mV at 143 mM [Na⁺]o, ?37 ± 3 mV at 1.2 mM [Na⁺]o), depolarization‐induced Ca²⁺ influx was not a cause of the low [Na⁺]o‐induced contraction. The A_MS did not develop significant myogenic tone. Although low [Na⁺]o also constricted A_MS, the magnitude of constriction was significantly weaker than that in A_SK (17 ± 4 vs. 47 ± 6%, P < 0.01, at 58 mM Na⁺). With Bay K 8644, A_MS developed myogenic tone, and low [Na⁺]o‐induced constriction was significantly increased. In conclusion, Na⁺/Ca²⁺ exchanger may play an important role in regulating myogenic tone, likely via mediating Ca²⁺‐extrusion.     

自发性高血压大鼠脑细小动脉神经肽Y分布的研究

为了解血管周围神经肽类分布与高血压时细小动脉构型重建之关系，我们采用自发性高血压大鼠（ＳＨＲ）软脑膜铺片技术，展示了血管网的各级分支，对血管系统进行了全面、立体的观察。并用免疫组织化学、图像分析仪对血管网上神经肽Ｙ（ＮＰＹ）分布进行定性定量分析。结果表明，ＳＨＲ大脑中动脉及其分支，小动脉、细动脉壁上含ＮＰＹ神经纤维分布表面积密度及ＮＰＹ抗体阳性反应物积分光密度均明显高于正常血压组（ＷＫＹ），这一实验结果揭示脑细小动脉含ＮＰＹ神经分布密度增加，使血管平滑肌营养调节作用增强，与其动脉收缩兴奋性增高，动脉平滑肌细胞肥大和高血压的发生发展有着明显关系。     

Neonatal pre-ganglionic sympathectomy affects morphometrically defined architecture in rat cerebral arteries

In order to clarify further the nature of the long-term influence on the cerebral vasculature by the sympathetic nerves, a bilateral cervical pre-ganglionic denervation was performed in 1-week-old rats. Four weeks later, morphometric determinations of the vascular dimensions revealed diminished media cross-sectional areas and luminal radii in the middle cerebral and posterior cerebral arteries, whereas these parameters were unaffected in the basilar artery. In the latter artery, however, a 40% increase in the cross-sectional area of the internal elastic membrane was found. No re-innervation of the denervated ganglia occurred during the course of the experiment. The results suggest that the long-term effect exerted by the sympathetic nerves is associated with the nerve activity, rather than being a true trophic influence.     

Development of neuromuscular junctions on small mesenteric arteries of the rat

This ultrastructural study has investigated the development of the innervation of second order mesenteric arteries from the ileum region of the rat intestine, particularly, the time course of the formation of the plexus of varicose axons around the arteries, and the formation of autonomic neuromuscular junctions. The time points studied were postnatal days-2, -4, -8 and -13. This study has revealed that the formation of neuromuscular junctions with mature structural characteristics occurred at ~2 weeks postnatal. The plexus of varicose axons developed predominantly between day-4 and day-13, which agrees with previous light microscopy studies of catecholamne containing nerves around similar vessels. At day-2 and day-4, the axons lacked varicosities and were mainly contained in large bundles located in the outer region of the adventitia. The medio-adventitial border consisted of a dense layer of extracellular matrix and fibroblasts. By day-8, there were more axons and most were distributed in smaller bundles. Some had grown through the adventitia to lie at the medio-adventitial border and axon varicosities were also observed. Some varicosities had formed rudimentary neuromuscular contacts. By day-13, there were significantly more contacting varicosities compared to day-8. They were structurally more mature, being twice the size with three times the number of synaptic vesicles and consistently contained a mitochondrion. Conversely, the neuromuscular contact areas were similar at both time points. Some organisation of the synaptic vesicles associated with the prejunctional membrane, was evident in varicosities at day-8 but there were no presynaptic membrane specialisations similar to the putative neurotransmitter release sites found at mature skeletal neuromuscular junctions. The aggregation of small vesicles at the prejunctional membrane was more pronounced in neuromuscular junctions at day-13 with some having presynaptic membrane specialisations. Comparison of the structure of developing autonomic neuromuscular junctions with that of skeletal neuromuscular junctions has revealed a number of similarities.     

Effects of selective phosphodiesterase inhibitors on isolated coronary,lung and renal arteries from man and rat

The relaxant effects of some isozyme-selective phosphodiesterase inhibitors were evaluated in isolated human and rat coronary, lung and renal arteries. Milrinone and OPC 3911, inhibitors of a cGMP-inhibited cAMP phosphodiesterase (cGI-PDE), were shown to have distinct vasodilator actions. These agents were less potent as relaxants in rat lung and renal arteries than in the corresponding human tissues. OPC 3911, the more potent cGMP-inhibited cAMP phosphodiesterase inhibitor, was found to be the more potent vasorelaxant. Rolipram, a selective inhibitor of a cGMP-noninhibited cAMP phosphodiesterase, had small effects on coronary and lung arteries, but produced a slightly more pronounced relaxation of renal arteries from both man and rat. In human preparations contracted by 30 mm K⁺, milrinone and OPC 3911 had similar relaxant profiles, and were as potent in coronary as in renal arteries. These results do not support the notion that milrinone has reduced effects on renal vessels in man and show that there may be species differences in vascular responsiveness to cGMP-inhibited cAMP phosphodiesterase inhibitors.     

Anatomical variations of the renal arteries and renal transplantation

This study reports a retrospective analysis of the details of the anatomy of the renal arteries of 403 kidney donors and the arterial complications related to 513 subsequent kidney transplants in our transplant unit. Multiple renal arteries occurred bilaterally in 10.2% of donors and unilaterally in 20.8%, a total incidence of 31%. There was a higher incidence of vascular-related complications following transplantation of kidneys with multiple renal arteries. Attention is drawn to the need for careful technique in identification of multiple renal vessels, especially aberrant vessels, at the time of donor nephrectomy and also to the different techniques available for anastomosis of multiple renal arteries in kidney transplant recipients. © 1992 Wiley-Liss, Inc.     

Sympathetic a-adrenergic control of large-bore arterial vessels,arterioles and veins,and of capillary pressure and fluid exchange in whole-organ cat skeletal muscle

The sympathetic nervous control of the vascular bed of cat gastrocnemius muscle was studied with a new whole-organ technique which permits simultaneous, continuous and quantitative measurements of capillary pressure (P_c), capillary fluid exchange and resistance reactions in the whole vascular bed and in its three consecutive sections: large-bore arterial vessels (> 25 μm), arterioles (< 25 μm) and veins. The results demonstrated a distinct neural control of all three consecutive vascular sections, graded in relation to the rate of nerve excitation up to maximum at 16 Hz. Stimulation at high rates, which in the steady state caused an average rise of overall regional resistance from 15.3 to 120 PRU (7.8-fold increase), thus raised large-bore arterial vessel resistance from 8.8 to 64 PRU (7.3-fold increase), arteriolar resistance from 4.5 to 49 PRU (10.9-fold increase) and venous resistance from 2.0 to 7 PRU (3.5-fold increase). The rate of resistance development (PRU s^-1) of the sympathetic constrictor response was much higher in the arteriolar than in the other sections, which indicates that the neural control is especially prompt and efficient in the arterioles. A passive component was shown to contribute to the described responses only on the venous side, but in no case by more than 10% of the total sympathetic venous resistance response, which thus is mainly active. Of special functional importance was that the new technique provided information about the adrenergic control of P_c, in absolute figures. From the control value of 19 mmHg, graded sympathetic stimulation caused a graded decline in P_c, at maximum constriction by about 7 mmHg. This resulted in marked net transcapillary fluid absorption, in turn increasing plasma volume.     

Effect of chronic electrical stimulation and β-GPA diet on GLUT4 protein concentration in rat skeletal muscle

The present study investigated whether alterations in the muscle high energy phosphate state initiates the contraction-induced increase in skeletal muscle GLUT4 protein concentration. Sprague-Dawley rats were provided either a normal or a 2% β-guanidinoproprionic acid (β-GPA) diet for 8 weeks and then the gastrocnemius of one hind limb was subjected to 0, 14 or 28 days of chronic (24 h day^?1) low-frequency electrical stimulation (10 Hz). The β-GPA diet, in the absence of electrical stimulation, significantly reduced ATP, creatine phosphate, creatine and inorganic phosphate and elevated GLUT4 protein concentration by 60% without altering adenylate cyclase activity or cAMP concentration. Following 14 days of electrical stimulation, GLUT4 protein concentration was elevated above non-stimulated muscle in both groups but was significantly more elevated in the β-GPA group. Concurrent with this greater rise in GLUT4 protein concentration was a greater decline in the high energy phosphates and a greater rise in cAMP. After 28 days of electrical stimulation, GLUT4 protein concentration and cAMP stabilized and was not different between diet treatments. However, the high energy phosphates were significantly higher in the normal diet rats as opposed to the β-GPA rats. These findings therefore suggest that a reduction in cellular energy supply initiates the contraction-induced increase in muscle GLUT4 protein concentration, but that a rise in cAMP may potentiate this effect.     

Invertebrate muscles: thin and thick filament structure; molecular basis of contraction and its regulation, catch and asynchronous muscle

This is the second in a series of canonical reviews on invertebrate muscle. We cover here thin and thick filament structure, the molecular basis of force generation and its regulation, and two special properties of some invertebrate muscle, catch and asynchronous muscle. Invertebrate thin filaments resemble vertebrate thin filaments, although helix structure and tropomyosin arrangement show small differences. Invertebrate thick filaments, alternatively, are very different from vertebrate striated thick filaments and show great variation within invertebrates. Part of this diversity stems from variation in paramyosin content, which is greatly increased in very large diameter invertebrate thick filaments. Other of it arises from relatively small changes in filament backbone structure, which results in filaments with grossly similar myosin head placements (rotating crowns of heads every 14.5nm) but large changes in detail (distances between heads in azimuthal registration varying from three to thousands of crowns). The lever arm basis of force generation is common to both vertebrates and invertebrates, and in some invertebrates this process is understood on the near atomic level. Invertebrate actomyosin is both thin (tropomyosin:troponin) and thick (primarily via direct Ca(++) binding to myosin) filament regulated, and most invertebrate muscles are dually regulated. These mechanisms are well understood on the molecular level, but the behavioral utility of dual regulation is less so. The phosphorylation state of the thick filament associated giant protein, twitchin, has been recently shown to be the molecular basis of catch. The molecular basis of the stretch activation underlying asynchronous muscle activity, however, remains unresolved.     

Rhythmic contractions of isolated small arteries from rat: role of calcium

In order to investigate the mechanisms behind rhythmic contractions in small arteries of the mesenteric arcade from Wistar rats, the calcium dependency of the oscillations in response to noradrenaline activation was tested on isolated vessels. Application of 1 μM ryanodine or 30 μM TMB-8 (procedures known to inhibit Ca²⁺ release from intracellular stores) totally abolished the rhythmic activity, even though the antagonists had opposite effects on the amplitude of the contractile response to noradrenaline. Verapamil (1 μ) or felodipine (1 μ M) (agents known to inhibit influx of extracellular Ca²⁺) also abolished the oscillations and reduced the maximal noradrenaline response by about 40%. Reducing the extracellular Ca²⁺ concentration to 0.1 DIM reduced the amplitude of the noradrenaline response to a similar extent as 1 nM felodipine, but did not eliminate the oscillations. This may indicate that the effect of calcium entry blockers was to eliminate the voltage-dependency of Ca²⁺ inflow rather than just reducing the Ca⁸⁺ level. Manoeuvres that would increase the cytosolic Ca²⁺ concentration (exposure to caffeine or to the calcium agonist BAY-K 8644) increased the frequency of the oscillations. These observations indicate an important role, not only for voltage-operating channels, but also for intracellular calcium stores in the generation of rhythmic contractions in these small arteries. Oscillations appear to be generated by an interplay between membrane activation and intracellular calcium stores.