Interaction between bradykinin and voltage-sensitive sodium channels in myelinated nerve fibers

Summary The effects of externally and internally applied bradykinin on the excitability of single myelinated nerve fibers were studied. External bradykinin (10 M) slightly prolongs the action potential of a single myelinated nerve fiber; hence, when the fibers are stimulated by long-lasting pulses, this raises the frequency of repetitive firing in sensory fibers and evokes repetitive activity in motor fibers. Under voltage-clamp conditions, sodium channel inactivation is slowed, while sodium channel activation remains unaffected. Prolonged depolarization of th membrane leads to a maintained sodium current. The voltage dependence of the steady-state sodium current inactivation (h ) is shifted in the depolarized direction by 10 mV. Internally applied bradykinin produces a frequency-dependent block of the sodium current. The phenomena described here imply that more than one site on the sodium channel is modified by bradykinin.     

Voltage-gated sodium channel-associated proteins and alternative mechanisms of inactivation and block

Voltage-gated sodium channels mediate inward current of action potentials upon membrane depolarization of excitable cells. The initial transient sodium current is restricted to milliseconds through three distinct channel-inactivating and blocking mechanisms. All pore-forming alpha subunits of sodium channels possess structural elements mediating fast inactivation upon depolarization and recovery within milliseconds upon membrane repolarization. Accessory subunits modulate fast inactivation dynamics, but these proteins can also limit current by contributing distinct inactivation and blocking particles. A-type isoforms of fibroblast growth factor homologous factors (FHFs) bear a particle that induces long-term channel inactivation, while sodium channel subunit Navβ4 employs a blocking particle that rapidly dissociates upon membrane repolarization to generate resurgent current. Despite their different physiological functions, the FHF and Navβ4 particles have similarity in amino acid composition and mechanisms for docking within sodium channels. The three competing channel-inactivating and blocking processes functionally interact to regulate a neuron’s intrinsic excitability.     

Potassium currents in cardiac cells

The kinetic properties of the inwardly rectifying K current and the transient outward current in cardiac cells were investigated. In sheep Purkinje fibers superfused with Na-free K-free solution, time-dependent changes in the conductance of the inward rectifier are described. In patch clamp experiments the inward rectifier inactivates during hyperpolarization, as can be seen by a decrease in the open state probability. Using whole cell clamp on ventricular myocytes it is demonstrated that the inactivation during hyperpolarization is due to blocking of the channel by external Na, Mg and Ca. The channels responsible for the transient outward current in cow, sheep and rabbit Purkinje fibers are identified using single channel recording. It is demonstrated that in all three preparations the channels are K-selective. The channel in cow Purkinje cells has a large conductance and is regulated by voltage and internal Ca concentration. The channels identified in the sheep and rabbit cells have a much smaller conductance.     

The conduction velocity, number, and diameter of unmyelinated fibers in Remak's nerve

Remak's nerve in the chicken was examined ultrastructurally and electrophysiologically to determine the characteristics of fibers in the nerve trunk. The ration of unmyelinated fibers to myelinated ones was 111:1. The mean number of unmyelinated fibers was 3555 plus/minus 232 (SEM, n=5) and they had a mean diameter of 0.502 plus/minus 0.034 (SEM) micron. The compound action potential consisted almost entirely of a large diphasic waveform which had a mean peak conduction velocity of 0.62 plus/minus 0.031 (SEM, n=5) m.s-1 at 37 degrees C.     

An analysis of the distribution of the myelinated nerve fibers in the optic fascicle of a beagle dog

The composition of both optic fascicles of a Beagle dog was studied in topographically oriented, semithin transections of whole nerve stained with toluidine blue. About 165,000 myelinated fibers were present in each nerve, their maximum caliber reaching 11 micron; large, less densely arranged fibers occurred especially in the dorso-temporal region.     

Dantrolene: evidence for effects on Na permeability properties of the nodal membrane

The effects of dantrolene on myelinated frog nerve fibers were studied in voltage clamp experiments. Dantrolene shifted the potential-dependent parameters describing Na+ permeability towards more negative membrane potentials. The findings are interpreted as a change in the negative surface charge of the membrane.     

Zur wirkung hypertoner Lösungen auf die markhaltige Nervenfaser

Summary The effect of hypertonic solutions on the action potential of single myelinated nerve fibres is described. Hypertonicity mainly changes the duration of the action potential: Short action potentials obtained in normal Ringer's solution at room temperature are prolonged, long action potentials due to 0.1–1.0 mM NiCl₂-Ringer's solution and low temperature are shortened by hypertonicity. The changes in action potential duration are accompanied by small changes in action potential amplitude. In addition, hypertonicity reduces the depolarization produced by 20 mM KCl; inactivation of the sodium-carrying system under cathodal polarization is enhanced.

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Die Untersuchungen wurden mit Unterstützung der Deutschen Forschungsgemeinschaft ausgeführt.     

The conduction velocity,number and diameter of unmyelinated fibers in Remak's nerve

Summary Remak's nerve in the chicken was examined ultrastructurally and electrophysiologically to determine the characteristics of fibers in the nerve trunk. The ratio of unmyelinated fibers to myelinated ones was 1111. The mean number of unmyelinated fibers was 3555±232 (SEM, n=5) and they had a mean diameter of 0.502±0.034 (SEM) m. The compound action potential consisted almost entirely of a large diphasic waveform which had a mean peak conduction velocity of 0.62±0.031 (SEM, n=5) m·s^–1 at 37°C.     

An analysis of the distribution of the myelinated nerve fibers in the optic fascicle of a Beagle dog

Summary The composition of both optic fascicles of a Beagle dog was studied in topographically oriented, semithin transections of whole nerve stained with toluidine blue. About 165,000 myelinated fibers were present in each nerve, their maximum caliber reaching 11 m; large, less densely arranged fibers occurred especially in the dorso-temporal region.We are grateful to Dr D.C. Naylor, Mrs K. Schnider, Mr F. da Silva and Mr K. Traber for their aid.     

Frequency-dependent effect of verapamil on rat soleus muscle

In the presence of verapamil (0.1 mM) rat soleus muscle fibers failed to generate action potentials with overshoots. In fibers with their Vm set to a local level of -90 mV, verapamil produces a gradual reduction in the amplitude of the repetitive action potentials; this effect is more pronounced at high rates of stimulation (100 Hz). Our results suggest a local anesthetic action of this drug that could contribute with its calcium channel blocking effect to the diminished mechanical tension observed in the presence of the drug.     

Periodic variations in motor endplate potentials]

The amplitudes of motor end-plate potentials in the Frog have been measured during repetitive stimulations of the motor nerve at frequencies between 0,5 and 30 Hz. A serial analysis showed that cyclic variations of these amplitudes were superimposed on the random fluctuation of epp due to the quantal nature of transmitter release. The period of these cyclic variations varied between 3 and 10 stimulations. This period was not significantly related to the frequency of the stimulation. The frequency spectrum analysis of the epp amplitudes showed similar values for the period of the cyclic variation of epp. The same kind of analysis applied to a binomial series of random numbers did not significantly present periodic fluctuations. The same analysis was applied to mean amplitudes of groups of 5 to 120 successive epps at the same end-plate. Periodic fluctuations were identified with periods of about 10 to 180 sec. It is suggested that periodic failures of nerve spike propagation in nerve terminals at sites of low safety factor, as at branchings, are responsible for the observed cyclic variation of epp.     

Inhibitory action of bradykinin on release of the adrenergic transmitter in the isolated lapine kidney

Summary In the isolated lapine kidney perfused with tyrode solution and prelabeled with [³H] norepinephrine, bradykinin (10 ng/ml) decreased the overflow of tritium elicited by sympathetic nerve stimulation both in the presence and absence of indomethacin. These observations indicate that bradykinin acts at presynaptic sites reducing release of the adrenergic transmitter in the isolated lapine kidney.This study was supported by American Heart Association Grant 77-803, and U.S. Public Health Service Grant HL-16134 from the National Heart and Lung Institute.     

Frequency-dependent effect of verapamil on rat soleus muscle

Summary In the presence of verapamil (0.1 mM) rat soleus muscle fibers failed to generate action potentials with overshoots. In fibers with their V_m set to a local level of –90 mV, verapamil produces a gradual reduction in the amplitude of the repetitive action potentials; this effect is more pronounced at high rates of stimulation (100 Hz). Our results suggest a local anesthetic action of this drug that could contribute with its calcium channel blocking effect to the diminished mechanical tension observed in the presence of the drug.Acknowledgments. We thank A. Losavio and M. Stefanolo for technical assistance. This work was supported by grants from CONICET and SUBCYT, Buenos Aires, Argentina and Muscular Dystrophy Association, USA.     

The effect of a bradykinin antagonist on vasodilator responses with particular reference to the submandibular gland of the cat

A bradykinin analogue, D-Arg[Hyp3, Thi5,8, D-Phe7]-Bk, antagonized the vasodilator effect of bradykinin injected close-arterially in the submandibular salivary gland of the cat, without affecting that due to acetylcholine or nerve stimulation. The same analogue also antagonized the hypotensive response to bradykinin injected intravenously in cats and rabbits. We conclude that functional hyperaemia in the submandibular gland of the cat is not due to the release of bradykinin by salivary kallikrein.     

Conditioning of depressor responses evoked by single volleys in the aortic nerve

Summary Depressor responses evoked by single volleys in the aortic nerve are more facilitated by conditioning volleys exciting C fibers in the same aortic nerve than in the contralateral one. Conditioning volleys in A fibers do not facilitate the testing depressor responses.     

Sodium channels in cardiac Purkinje cells

Sodium (Na+) currents are responsible for excitation and conduction in most cardiac cells, but their study has been hampered by the lack of a satisfactory method for voltage clamp. We report a new method for low resistance access to single freshly isolated canine cardiac Purkinje cells that permits good control of voltage and intracellular ionic solutions. The series resistance was usually less than 3 omega cm2, similar to that of the squid giant axon. Cardiac Na+ currents resemble those of nerve. However, Na+ current decay is multiexponential. The basis for this was further studied with cell-attached patch clamp recording of single Na+ channel properties. A prominent characteristic of the single channels was their ability to reopen after closure. There was also a long opening state that may be the basis for a small very slowly decaying Na+ current. This rare long opening state may contribute to the Na+ current during the action potential plateau.     

Myelination in the hippocampus during development and following lesion

Myelin is crucial for the stabilization of axonal projections in the developing and adult mammalian brain. However, myelin components also act as a non-permissive and repellent substrate for outgrowing axons. Therefore, one major factor which accounts for the lack of axonal regeneration in the mature brain is myelin. Here we report on the appearance of mature, fully myelinated axons during hippocampal development and following entorhinal lesion with the myelin-specific marker Black Gold. Although entorhinal axons enter the hippocampal formation at embryonic day 17, light and ultrastructural analysis revealed that mature myelinated fibers in the hippocampus occur in the second postnatal week. During postnatal development, increasing numbers of myelinated fibers appear and the distribution of myelinated fibers at postnatal day 25 was similar to that found in the adult. After entorhinal cortex lesion, a specific anterograde denervation in the hippocampus takes place, accompanied by a long-lasting loss of myelin. Quantitative analysis of myelin and myelin breakdown products at different time points after lesion revealed a temporally close correlation to the degeneration and reorganization pha-ses in the hippocampus. In contrast, electroconvulsive seizures resulted in brief demyelination and a faster recovery time course. In conclusion, we could show that the appearance of mature axons in the hippocampus is temporally regulated during development. In the adult hippocampus, demyelination was found after anterograde degeneration and also following seizures, suggesting that independent types of insult lead to demyelination. Reappearing mature axons were found in the hippocampus following axonal sprouting. Therefore, our quantitative analysis of mature axons and myelination effectively reflects the readjusted axonal density and possible electrophysiological balance following lesion. Received 22 December 2003; received after revision 11 February 2004; accepted 17 February 2004     

Patch and whole-cell voltage clamp of single mammalian visceral and vascular smooth muscle cells

Dispersal of the constituent cells of mammalian visceral and vascular smooth muscles has permitted recordings both of membrane currents under whole-cell voltage clamp, and of currents through single ionic channels using the patch-clamp technique. A rectangular depolarizing step applied to a single cell under voltage clamp yielded a net inward current followed by a net outward current in normal physiological solution. In isolated, 'inside-out' patches of cell membrane a calcium- and potential-sensitive K channel (100 pS conductance) and a calcium-insensitive, potential-sensitive K+ channel (50 pS conductance) with slow kinetics have so far been identified and characterized.     

The effect of a bradykinin antagonist on vasodilator responses with particular reference to the submandibular gland of the cat

Summary A bradykinin analogue, D-Arg[Hyp³, Thi^{5, 8}, D-Phe⁷]-Bk, antagonized the vasodilator effect of bradykinin injected close-arterially in the submandibular salivary gland of the cat, without affecting that due to acetylcholine or nerve stimulation. The same analogue also antagonized the hypotensive response to bradykinin injected intravenously in cats and rabbits. We conclude that functional hyperaemia in the submandibular gland of the cat is not due to the release of bradykinin by salivary kallikrein.     

Patch and whole-cell voltage clamp of single mammalian visceral and vascular smooth muscle cells

Summary Dispersal of the constituent cells of mammalian visceral and vascular smooth muscles has permitted recordings both of membrane currents under whole-cell voltage clamp, and of currents through single ionic channels using the patch-clamp technique. A rectangular depolarizing step applied to a single cell under voltage clamp yielded a net inward current followed by a net outward current in normal physiological solution. In isolated, inside-out patches of cell membrane a calcium- and potential-sensitive K channel (100 pS conductance) and a calcium-insensitive, potential-sensitive K⁺ channel (50 pS conductance) with slow kinetics have so far been identified and characterized.