Neural and humoral control of regional vascular beds via A1 adenosine receptors located in the nucleus tractus solitarii

Our previous studies showed that stimulation of adenosine A(1) receptors located in the nucleus of the solitary tract (NTS) exerts counteracting effects on the iliac vascular bed: activation of the adrenal medulla and β-adrenergic vasodilation vs. sympathetic and vasopressinergic vasoconstriction. Because NTS A(1) adenosine receptors inhibit baroreflex transmission in the NTS and contribute to the pressor component of the HDR, we hypothesized that these receptors also contribute to the redistribution of blood from the visceral to the muscle vasculature via prevailing sympathetic and vasopressinergic vasoconstriction in the visceral (renal and mesenteric) vascular beds and prevailing β-adrenergic vasodilation in the somatic (iliac) vasculature. To test this hypothesis, we compared the A(1) adenosine-receptor-mediated effects of each vasoactive factor triggered by NTS A(1) adenosine receptor stimulation [N(6)-cyclopentyladenosine (CPA), 330 pmol in 50 nl] on the regional vascular responses in urethane/chloralose-anesthetized rats. The single-factor effects were separated using adrenalectomy, β-adrenergic blockade, V(1) vasopressin receptor blockade, and sinoaortic denervation. In intact animals, initial vasodilation was followed by large, sustained vasoconstriction with smaller responses observed in renal vs. mesenteric and iliac vascular beds. The initial β-adrenergic vasodilation prevailed in the iliac vs. mesenteric and renal vasculature. The large and sustained vasopressinergic vasoconstriction was similar in all vascular beds. Small sympathetic vasoconstriction was observed only in the iliac vasculature in this setting. We conclude that, although A(1) adenosine-receptor-mediated β-adrenergic vasodilation may contribute to the redistribution of blood from the visceral to the muscle vasculature, this effect is overridden by sympathetic and vasopressinergic vasoconstriction.     

Three different vasoactive responses of rat tail artery to nicotine

The vasoactive effects of nicotine on isolated rat tail artery tissues were studied. Nicotine transiently contracted rat tail artery tissues (EC50, 55.6 +/- 2 microM) in an extracellular Ca2+ dependent and endothelium-independent fashion. The blockade of alpha1-adrenoceptors, but not alpha2-adrenoceptors or P2X purinoceptors, inhibited the nicotine-induced contraction by 38 +/- 7% (p < 0.05). Nicotine (1 mM) depolarized membrane by 13 +/- 3 mV, but did not affect L-type Ca2+ channel currents, of the isolated rat tail artery smooth muscle cells. The phenylephrine-precontracted tail artery tissues were relaxed by nicotine (EC50, 0.90 +/- 0.31 mM), which was significantly inhibited after the blockade of nicotinic receptors. Simultaneous removal of phenylephrine and nicotine, after a complete relaxation of the phenylephrine-precontracted tail artery strips was achieved by nicotine at accumulated concentrations (> or =10 mM), triggered a Ca2+-dependent rebound long-lasting vasoconstriction (n = 20). This rebound contraction was abolished in the absence of calcium or in the presence of tetracaine in the bath solution. Pretreatment of vascular tissues with a nicotinic receptor antagonist did not affect the nicotine-induced vasoconstriction or nicotine withdrawal induced rebound contraction. The elucidation of the triphasic vascular effects of nicotine and the underlying mechanisms is important for a better understanding of the complex vascular actions of nicotine.     

Stimulation of NTS A1 adenosine receptors evokes counteracting effects on hindlimb vasculature

Our previous studies concluded that stimulation of the nucleus of the solitary tract (NTS) A2a receptors evokes preferential hindlimb vasodilation mainly via inducing increases in preganglionic sympathetic nerve activity (pre-ASNA) directed to the adrenal medulla. This increase in pre-ASNA causes the release of epinephrine and subsequent activation of beta-adrenergic receptors that are preferentially located in the skeletal muscle vasculature. Selective activation of NTS A1 adenosine receptors evokes variable, mostly pressor effects and increases pre-ASNA, as well as lumbar sympathetic activity, which is directed to the hindlimb. These counteracting factors may have opposite effects on the hindlimb vasculature resulting in mixed vascular responses. Therefore, in chloralose-urethane-anesthetized rats, we evaluated the contribution of vasodilator versus vasoconstrictor effects of stimulation of NTS A1 receptors on the hindlimb vasculature. We compared the changes in iliac vascular conductance evoked by microinejctions into the NTS of the selective A1 receptor agonist N6-cyclopentyladenosine (330 pmol in 50 nl volume) in intact animals with the responses evoked after beta-adrenergic blockade, bilateral adrenalectomy, bilateral lumbar sympathectomy, and combined adrenalectomy + lumbar sympathectomy. In intact animals, stimulation of NTS A1 receptors evoked variable effects: increases and decreases in mean arterial pressure and iliac conductance with prevailing pressor and vasoconstrictor effects. Peripheral beta-adrenergic receptor blockade and bilateral adrenalectomy eliminated the depressor component of the responses, markedly potentiated iliac vasoconstriction, and tended to increase the pressor responses. Lumbar sympathectomy tended to decrease the pressor and vasoconstrictor responses. After bilateral adrenalectomy plus lumbar sympathectomy, a marked vasoconstriction in iliac vascular bed still persisted, suggesting that the vasoconstrictor component of the response to stimulation of NTS A1 receptors is mediated mostly via circulating factors (e.g., vasopressin, angiotensin II, or circulating catecholamines released from other sympathetic terminals). These data strongly suggest that stimulation of NTS A1 receptors exerts counteracting effects on the iliac vascular bed: activation of the adrenal medulla and beta-adrenergic vasodilation versus vasoconstriction mediated by neural and humoral factors.     

Lipoprotein lipase activity in skeletal muscles of the rat: effects of denervation and tenotomy.

The effects of denervation, tenotomy, or tenotomy with simultaneous denervation on the activity of heparin-releasable and intracellular, residual lipoprotein lipase (LPL) and triacylglycerol (TG) content were examined in rat skeletal muscles. An influence of muscle electrostimulation on denervated and tenotomized muscles was also evaluated. Activity of both LPL fractions was decreased in denervated and/or tenotomized soleus and red portion of gastrocnemius muscles. It was accompanied by a slight elevation of the intracellular TG content. Electrostimulation increased activities of both fractions of LPL in red muscles from intact hindlimbs. In stimulated denervated muscles without or with simultaneous tenotomy, activity of two LPL fractions was also enhanced, but control values were reached only in denervated soleus muscle. Electrical stimulation had no pronounced effect on LPL activity in tenotomized muscles. In conclusion, denervation and/or tenotomy decreases LPL activity in red muscles, indicating reduction of the muscle potential to utilize circulating TG. Electrostimulation only partly restores the diminished LPL activity in denervated muscles, without any effect in tenotomized ones. Thus, to maintain LPL activity in resting muscle, intact innervation and tension are needed.     

Mechanisms mediating canine renal vasoconstriction induced by nicotine infusion

We investigated the respective contributions of the renin-angiotensin and alpha-adrenergic systems to nicotine-induced, canine, renal vasoconstriction by using saralasin (4 micrograms/kg/min) and phentolamine (25 micrograms/kg/min) blockade respectively. Nicotine infusion (0.024 mg/kg/min) increased mean arterial blood pressure (MABP) (114 +/- 3.0 to 219 +/- 8.0 mmHg) and decreased total renal blood flow (TRBF) (3.12 +/- 0.34 to 1.60 +/- 0.37 ml/min/g). Nicotine infusion produced a significantly lesser blood flow in outer cortex (OC), inner cortex (IC), and outer medulla (OM) compared to control dogs. The intrarenal-artery infusion of saralasin or phentolamine had no effect on the nicotine-induced MABP changes. Phentolamine infusion prior to nicotine resulted in a significantly greater TRBF (P less than 0.01), OC (p less than 0.001), IC (p less than 0.001) and OM (p less than 0.01) flow than in the group that received nicotine only. Saralasin pretreatment prior to nicotine resulted only in a significantly (p less than 0.01) greater OC flow than nicotine only. Our data suggest that while angiotensin II mediates a portion of the action of nicotine on the OC renal vasculature, the alpha adrenergic system predominates as the mediator of nicotine-induced renal vasoconstriction in the first 7 minutes of nicotine infusion.     

Combined NO and PG inhibition augments alpha-adrenergic vasoconstriction in contracting human skeletal muscle

Sympathetic alpha-adrenergic vasoconstrictor responses are blunted in the vascular beds of contracting muscle (functional sympatholysis). We tested the hypothesis that combined inhibition of nitric oxide (NO) and prostaglandins (PGs) restores sympathetic vasoconstriction in contracting human muscle. We measured forearm blood flow via Doppler ultrasound and calculated the reduction in forearm vascular conductance in response to alpha-adrenergic receptor stimulation during rhythmic handgrip exercise (6.4 kg) and during a control nonexercise vasodilator condition (using intra-arterial adenosine) before and after combined local inhibition of NO synthase (NOS; via N(G)-nitro-L-arginine methyl ester) and cyclooxygenase (via ketorolac) in healthy men. Before combined inhibition of NO and PGs, the forearm vasoconstrictor responses to intra-arterial tyramine (which evoked endogenous noradrenaline release), phenylephrine (a selective alpha1-agonist), and clonidine (an alpha2-agonist) were significantly blunted during exercise compared with adenosine treatment. After combined inhibition of NO and PGs, the vasoconstrictor responses to all alpha-adrenergic receptor stimuli were augmented by approximately 10% in contracting muscle (P <0.05), whereas the responses to phenylephrine and clonidine were also augmented by approximately 10% during passive vasodilation in resting muscle (P <0.05). In six additional subjects, PG inhibition alone did not alter the vasoconstrictor responses in resting or contracting muscles. Thus in light of our previous findings, it appears that inhibition of either NO or PGs alone does not affect functional sympatholysis in healthy humans. However, the results from the present study indicate that combined inhibition of NO and PGs augments alpha-adrenergic vasoconstriction in contracting muscle but does not completely restore the vasoconstrictor responses compared with those observed during passive vasodilation in resting muscle.     

Influence of alpha and beta adrenergic receptors blockade on the adrenolytic effect of muscular work

The changes in the response of adrenergic receptors alpha and beta in the blood vessels in the working muscles in a hindlimb in cats were studied after intra-arterial administration of noradrenaline, isoprenaline and during electric stimulation of the sympathetic trunk. The experiments were carried out during alpha-adrenergic receptors blockade with dihydroergotamine (0.3 mg/kg) beta-adrenergic receptors blockade with propranolol (1 mg/kg) and blockade of acetylcholine M receptors with atropine (0.5 mg/kg). The investigations were performed at rest, during exercise (electric stimulation of the sciatic nerve) and after the exercise. The following results deserve attention: 1) beta-adrenergic receptors blockade reduced significantly the alpha-adrenolytic effect of exercise restoring the ability of blood vessel to constriction in response to noradrenaline; 2) the vasodilator effect of isoprenaline evident in resting state and maintained to some extent during exercise was abolished completely by preceding alpha-adrenergic blockade. The changes in the reactivity of resistance vessels in working skeletal muscles to noradrenaline, with abolition of its vasoconstrictor effect, have been shown by Rein [7] and others authors [2, 5]. Similarly, it is well known that the resistance vessels contain two types of adrenergic receptors alpha and beta, and that the response of the vessels to stimulation of these receptors are different [1]. In view of the recently published observations of Jarhult and Lundvall suggesting that the beta-adrenergic receptors play an important physiological role [6] in the arterial part of the microcirculation [6] and in view of the hypothesis put forward by Kunos and Szentivanyj that alpha and beta receptors can be transformed depending on the intensity of tissue metabolism [8] it seemed worth while to study more systematically the changes of the reactivity of alpha and beta adrenergic receptors in the vascular bed of the skeletal muscles during and after muscle exercise.     

Protease Inhibitors Reduce Effects of Denervation on Muscle End-Plate Acetylcholinesterase

The effects of certain protease inhibitors on end-plate acetylcholinesterase (AChE) activity, as well as on wet weight and total protein, were studied in vivo in intact and denervated anterior gracilis muscles from the rat. A combination of leupeptin, pepstatin, and aprotinin, administered intraarterially, partly prevented the early (24 h) denervation-induced decrease in muscle weight and protein content. In turn, leupeptin and aprotinin, either alone or in combination, markedly reduced the decay of AChE activity in the denervated muscles, whereas pepstatin alone was ineffective. Such effects were additive in that the inhibitors in combination were more effective than when they were used separately. Additional experiments indicated that none of the inhibitors, at the concentrations used, affected AChE activity directly, nor did they have a significant effect during processing of the muscle samples. These findings indicate that the initial decay of AChE activity with denervation was effectively reduced by the inhibitors, probably through inactivation of proteolytic enzymes which, otherwise, would be increase in denervated muscle.     

Changes in vascular responsiveness following chronic exposure to cold in the rat

The responsiveness of smooth muscle from rings of aortic tissue of cold-acclimated (CA, 6 degrees C, 5-15 wk) rats to both alpha- and beta-adrenergic agonists and KCl was tested and compared with that of warm-adapted (25 degrees C) controls. alpha-Adrenergic stimulation, induced by low doses (10(-8)-10(-7) M) of phenylephrine and norepinephrine in the presence and absence of the beta-adrenergic antagonist, propranolol, resulted in the development of less active tension by aortic smooth muscle from CA rats than from controls. Similar results were observed with the weakly alpha 1-adrenergic agonistic activities of tyramine, clonidine, and high concentrations of isoproterenol (10(-6)-10(-4) M). There was also a significant reduction in the tension developed by smooth muscles of the aortas from CA rats when depolarized with KCl in concentrations ranging from 8 to 20 mM. In contrast, aortic smooth muscle, contracted to 75% of maximum with KCl, showed an enhanced relaxation to the beta-adrenergic agonist, isoproterenol, in CA rats. These studies suggest that acclimation of rats to cold results in both a decrease in alpha-adrenergic responsiveness and an increase in beta-adrenergic responsiveness in vascular smooth muscle as well as a change in the biochemical events that couple activation of adrenergic receptors to changes in vasomotor tone.     

Carbon monoxide promotes endothelium-dependent constriction of isolated gracilis muscle arterioles

Vascular tissues express heme oxygenase, which metabolizes heme to form carbon monoxide (CO). CO promotes relaxation of vascular smooth muscle but also inhibits nitric oxide (NO) formation. This study examines the hypothesis that CO promotes endothelium- and NO synthase-dependent vasoconstriction of isolated arterioles. Studies were conducted on pressurized first-order gracilis muscle arterioles isolated from anesthetized male Sprague-Dawley rats. Exogenous CO, as well as a heme precursor, delta-aminolevulinic acid (delta-ALA), constricted arterioles with intact endothelium pretreated with phenylephrine; these effects were abolished by removal of the endothelium. CO- and delta-ALA-induced vasoconstrictions were converted to dilations by pretreatment with an inhibitor of NO synthase, Nomega-nitro-l-arginine methyl ester, or with Nomega-nitro-l-arginine methyl ester and an NO donor, sodium nitroprusside. Furthermore, CO-induced vasoconstriction was prevented by pretreatment with the NO synthase substrate l-arginine. This study shows that exogenous, as well as endogenously formed, CO can promote endothelium-dependent vasoconstriction in isolated gracilis muscle arterioles. Because CO-induced vasoconstriction is abolished by NO synthase blockade and by l-arginine, CO most likely promotes endothelium-dependent vasoconstriction by inhibiting endothelial NO formation.     

Alpha-adrenergic tone in the coronary circulation of the conscious dog

To examine the role of neural factors in the control of coronary vasoactivity in conscious animals, dogs were supplied with miniature pressure gauges in the aorta and left ventricle (to measure aortic and left ventricular pressures, respectively and with a flow probe on the left circumflex coronary artery (to measure coronary blood flow). The experiments were conducted several weeks after recovery from operation. Stimulation of the carotid chemoreceptor and pulmonary inflation elicited a biphasic reflex response. Initially, coronary vasodilation was observed; coronary blood flow tripled even after changes in metabolic factors were minimized by pretreatment with propranolol. A similar response occurred after a spontaneous deep breath. The coronary vasodilation could be blocked by alpha-adrenergic receptor blockade. The second phase of the response involved an increase in coronary vascular resistance, associated with elevated arterial pressure and an absolute reduction in coronary blood flow and coronary sinus oxygen content. The secondary coronary vasoconstriction was also abolished by alpha-adrenergic blockade. Paradoxically, alpha-adrenergic receptor blockade with phentolamine (at constant heart rate and after beta-adrenergic receptor blockade) did not increase coronary blood flow and reduced coronary vascular resistance only slightly. Selective alpha 1-adrenergic receptor blockade with prazosin and trimazosin on different days induced progressively greater reductions in coronary vascular resistance. Trimazosin was the only alpha-adrenergic receptor blocker to elevate coronary blood flow significantly. It is conceivable, but speculative, that withdrawal of alpha-adrenergic tone may involve activation of an intermediate agent, which is a potent coronary vasodilator. Alternatively, withdrawal of alpha-adrenergic tone may be an important mechanism for immediate control of the coronary circulation, but under more chronic conditions it plays a lesser role as a result of suppression by metabolic factors.     

Blockade of alpha-adrenergic receptors by analogues of phosphatidylcholine

The experimental evidence reviewed in this article suggests that the kidneys may have an additional function in regulating blood pressure besides their role in controlling both blood volume by urine formation and the relative state of vasoconstriction by the renin-angiotensin system. That is, the kidneys may have an additional influence upon the vasculature of a hormonal vasodilating system. The interstitial cells of the renal medulla appear to be mediating this activity and lipid compounds have been extracted from the renal medulla which display depressor activity. One such compound, the antihypertensive polar renomedullary lipid (APRL), has been demonstrated to consist of specific alkyl ether analogues of phosphatidylcholine. The vascular responses to these compounds include vasodilation of both arterioles and venules, rapid lowering of arterial blood pressure with little or no tachycardia, increased depressor activity in hypertensive animals, and blockade of vascular smooth muscle alpha 1-adrenergic receptors. Most recently, APRL and a synthetic analogue, 1-0-octadecyl-2-acetyl-sn-glycero-3-phosphorylcholine, have been used to demonstrate alpha-adrenergic receptor blockade on a smooth muscle cell line (DDT1) by radioligand assays. This action may be due to the insertion of these compounds into cell membranes causing subsequent steric interactions and blockade of the alpha-adrenergic receptor.     

Effect of denervation and phentolamine on the thermogenic action of noradrenaline in rat skeletal muscle

Vascularly isolated skeletal muscle of the cold-acclimated (CA) rat was perfused with blood in situ or in vitro and the effect of denervation and an alpha-adrenolytic agent (phentolamine) on its oxygen consumption was studied in the resting state and after administering noradrenaline (NA). The resting metabolism of muscle in situ rose by 28% after denervation. The infusion of NA further raised the oxygen consumption of acutely denervated muscle perfused in situ of in vitro by 43%. The thermogenic effect of NA on muscle denervated two hours before the experiment was only transitory. Phentolamine raised the oxygen consumption of the innervated muscle in situ by 42%; the infusion of NA did not stimulate metabolism any further. Phentolamine reduced the vascular resistance of resting muscle, but did not inhibit the vasoconstriction during the infusion of NA. The results show that the thermogenic effect of infused NA in perfused muscle is inhibited not by acute denervation, but by a vasoconstriction, which cannot be prevented by the administration of an alpha-adrenolytic agent.     

Acute systemic hypoxia elevates venous but not interstitial potassium of dog skeletal muscle

Potassium release through ATP-sensitive potassium (K(ATP)) channels contributes to hypoxic vasodilation in the skeletal muscle vascular bed: It is uncertain whether K(ATP) channels on muscle cells contribute to the process. Potassium from muscle cells must cross the interstitial space to reach the vascular tissues, whereas that from vascular endothelium would have a higher concentration in venous blood than in interstitial fluid. We determined the effect of systemic hypoxia on arterial, venous, and interstitial potassium in the constant-flow-perfused gracilis muscles of anesthetized dogs. Hypoxia reduced arterial Po(2) from 138 to 25 and Pco(2) from 28 to 26 mmHg. Arterial pH and potassium were well correlated (r(2) = 0.9): Both increased in early hypoxia and decreased during the postcontrol. In denervated muscles, perfusion pressure decreased from 95 to 76 mmHg by the end of the hypoxic period; neither venous nor interstitial potassium was elevated. In innervated muscles, perfusion pressure increased from 110 to 172 mmHg by the 11th min of hypoxia and then decreased to 146 mmHg by the end of the hypoxic period; venous potassium increased from 5.0 to 5.3 mM, but interstitial potassium remained unchanged. Glibenclamide abolished both the increase in venous potassium and the hypoxic vasodilation in the innervated muscle. Thus skeletal muscle cells were unlikely to have contributed to the release of potassium, which was suggested to originate from vascular endothelium. The sympathetic nerve supply may play a direct or indirect role in the opening of K(ATP) channels under hypoxic conditions.     

Systemic alpha-adrenergic and nitric oxide inhibition on basal limb blood flow: effects of endurance training in middle-aged and older adults

Endurance training improves endothelium-dependent vasodilation, yet it does not increase basal blood flow in the legs. We determined the effects of a 3-mo aerobic exercise intervention on basal leg blood flow and alpha-adrenergic vasoconstriction and nitric oxide (NO) release in seven apparently healthy middle-aged and older adults (60 +/- 3 yr). Basal femoral artery blood flow (via Doppler ultrasound) (pretraining: 354 +/- 29; posttraining: 335 +/- 34 ml/min) and vascular conductance did not change significantly with the exercise training. Before the exercise intervention, femoral artery blood flow increased 32 +/- 16% with systemic alpha-adrenergic blockade (with phentolamine) (P < 0.05), and the addition of nitric oxide synthase (NOS) inhibition using N(G)-monomethyl-L-arginine (L-NMMA) did not affect femoral artery blood flow. After training was completed, femoral artery blood flow increased 47 +/- 7% with alpha-adrenergic blockade (P < 0.01) and then decreased 18 +/- 7% with the subsequent administration of L-NMMA (P < 0.05). Leg vascular conductance showed a greater alpha-adrenergic blockade-induced vasodilation (+1.7 +/- 0.5 to +3.0 +/- 0.5 units, P < 0.05) as well as NOS inhibition-induced vasoconstriction (-0.8 +/- 0.4 to -2.7 +/- 0.7 units, P < 0.05) after the exercise intervention. Resting plasma norepinephrine concentration significantly increased after the training. These results suggest that regular aerobic exercise training enhances NO bioavailability in middle-aged and older adults and that basal limb blood flow does not change with exercise training because of the contrasting influences of sympathetic nervous system activity and endothelium-derived vasodilation on the vasculature.     

Role of different proteolytic systems in the degradation of muscle proteins during denervation atrophy

In order to clarify the cellular mechanisms of denervation atrophy of skeletal muscle, we have studied protein turnover in denervated and control rat soleus muscles in vitro under different conditions. By 24 h after cutting the sciatic nerve, overall protein breakdown was greater in the denervated soleus than in the contralateral control muscle, and by 3 days, net proteolysis had increased about 3-fold. Since protein synthesis increased slightly following denervation, the rise in proteolysis must be responsible for the muscle atrophy and the differential loss of contractile proteins. Like overall proteolysis, the breakdown of actin (as shown by 3-methyl-histidine production by the muscles) increased each day after denervation and by 3 days was 2.5 times faster than in controls. Treatments that block the lysosomal and Ca2(+)-dependent proteolytic systems did not reduce the increase in overall protein degradation and actin breakdown in the denervated muscles (maintained in complete medium at resting length). However, the content of the lysosomal protease, cathepsin B, increased about 2-fold by 3 days after denervation. Furthermore, conditions that activate intralysosomal proteolysis (incubation without insulin or amino acids) stimulated proteolysis 2-3-fold more in the denervated muscles than in controls. Also, incubation conditions that activate the Ca2(+)-dependent pathway (incubation with Ca2+ ionophores or allowing muscles to shorten) were 2-3 times more effective in enhancing overall proteolysis in the denervated muscle. None of these treatments affected 3-methylhistidine production. Thus, multiple proteolytic systems increase in parallel in the denervated muscle, but a nonlysosomal process (independent of Ca2+) appears mainly responsible for the rapid loss of cell proteins, especially of myofibrillar components.     

Neurotrophic control of 16S acetylcholinesterase from mammalian skeletal muscle in organ culture

The effects of rat obturator nerve extracts on total and 16S acetylcholinesterase (AChE) activity were studied in endplate regions of denervated anterior gracilis muscles maintained in organ culture for 48 hr. The decrease of total AChE activity in cultured muscles was similar to that observed in denervated muscles in vivo. This decrease in activity was partly prevented by addition of either 100 or 200 μl nerve extract (2.7 mg/ml protein) to the nutrient medium. Nerve extract treatment also decreased the release of AChE activity from the muscle into the bathing medium. Conversely, rat serum (20 μl; 90 mg/ml protein) had no effect on total AChE activity in muscle endplates, nor on release of the enzyme by the muscle. The 16S form of AChE was confined to motor endplate muscle regions and its activity was drastically decreased by denervation in both organ culture and in vivo preparations in a comparable manner. Nerve-extract supplemented cultures contained a significantly (p ? 0.001) larger amount of endplate 16S AChE activity (140–145%) than the corresponding controls (100-). Our results suggest that some nerve soluble substance, other than serum contaminants or 16S AChE itself, affects the maintenance of 16S AChE at the neuromuscular junction.     

Sustained cell proliferation in denervated skeletal muscle of mice

Summary Cellular proliferation in skeletal muscle was measured throughout the first 4 weeks after denervation. Twenty four mice had one leg denervated, and 4 groups of 6 of these mice were injected with tritiated thymidine once daily for 7 days, either during the first, second, third or fourth week after denervation. Autoradiographic labelling of muscle and connective tissue nuclei in denervated muscles was compared with innervated muscles from the opposite innervated legs of the same mice. Labelling of connective tissue and muscle (myonuclear and satellite cell) nuclei was significantly higher in denervated muscles, compared with innervated muscles on the unoperated side. There were no significant differences among labelling of nuclei in muscles denervated for 1, 2, 3 or 4 weeks. However, connective tissue labelling after 1 week of denervation was significantly higher than at later times. This study shows that nuclei of muscle and connective tissue cells proliferate and turnover at high levels for at least one month after denervation.     

Effects of sensitization of the guinea pig slow muscle in disorders of the neurotrophic control]

The immunohistochemical profile of intact and denervated soleus muscle of guinea pigs after sensibilization was studied. It is shown, that intact soleus muscle consists of slow fibers, which have low ATP-ase activity and don't react with monoclonal antibodies against fast myosin heavy chain. No changes of immunohistochemical profile were found after denervation or sensibilization. At the same time, the fibers, reacting with monoclonal antibodies against fast myosin heavy chain and having low ATP-ase activity, were found in denervated muscles after sensibilization. It is concluded, that the synthesis of fast myosin is induced after sensibilization of denervated muscles. Validity of myosin ATP-ase histochemistry for muscle fibers typing is discussed.