Calcium and cyclic AMP as possible mediators of neurohormone action in the hindgut of the cockroach, Leucophaea maderae

Adenosine 3′,5′-monophosphate (cyclic AMP) (10⁻⁵ g/ml) often caused a gradual increase in spotaneous contractile activity of the hindgut of the cockroach, Leucophaea maderae, and on rare occasions it would evoke a hormone-like response. However, aminophylline (2·5 × 10⁻⁴ g/ml) was capable of mimicking the neurohormone, and a concentration of 2·5 × 10⁻⁵ g/ml potentiated the contractile response evoked by the neurohormone: these responses were blocked by either the presence of 1 mM manganous ion or in a high potassium solution (162 mM). Propranolol (10⁻⁶ g/ml) and dopamine (10⁻⁴ g/ml) suppressed both spontaneous contractile events and neurohormone action. Dopamine (5 × 10⁻⁶ g/ml) also blocked action potential generation as did propranolol at 10⁻⁴ g/ml.These results lead us to suppose that cyclic AMP might serve as a mediator of neurohormone action by increasing calcium transport across the surface membrane of muscle fibres. Caffeine (2·5 × 10⁻⁴ g/ml), like aminophylline, caused a hormone-like response in normal hindguts. Even when the visceral muscles of the hindgut were depolarized in 162 mM potassium solution (without calcium), caffeine was still capable of inducing a phasic response. However, the addition of 2 mM calcium to such potassium-depolarized preparations caused a gradual increase in muscle tonus and substantially potentiated the response to caffeine.Such findings clearly implicate calcium as the mediator of excitation-contraction coupling in visceral muscle. While the interactions between the neurohormone, cyclic AMP, and calcium seem to be largely associated with the surface membrane and action potential generation.     

Visceral muscles and myogenic activity in the hindgut of the cockroach,Leucophaea maderae

Summary The hindgut of the Madeira cockroach contains an intricate network of longitudinal and circular muscles that are distinctive for each region. In the rectum, the longitudinal muscles are symmetrically arranged in 6 distinct bands, while the circular muscles appear as a uniform layer over the rectal pads. In the colon, the muscle fibers are arranged in an irregular lattice with the longitudinal fibers generally superimposed on the circular ones but with an evident weaving between the layers. In addition to these muscle layers, a delicate, superficial network of muscle-like fibers covers many portions of the colon and rectum.In spite of the bewilderingly complex motile activity of deganglionated hindguts, all activity could be classified under 4 basic types after cinematographic analysis: segmentation, compression, peristalsis, or reverse peristalsis or a combination thereof. Although much of the activity that occurred was seemingly random, there was an evident rhythmicity that spontaneously arose and ended in several types of motility during the course of observations. The defined modes of activity seemed to be completely myogenic in nature, as all 4 categories were readily observed in hindguts 30 min after treatment with tetrodotoxin (10^–6 g/ml). Each region of the hindgut seemed to have its own particular rhythm.Action potentials were recorded both intracellularly and extracellularly from all regions of the hindgut; amplitude usually ranged between 10 and 20 mV for intracellular recordings, and such spike potentials were often preceded by a slow depolarizing pre-potential. Generally, however, the depolarization was abrupt. Transmembrane potentials from the visceral muscle fibers were never truly at rest. Slow, continuous fluctuations (3–8 mV) were common. At times, plateau-type action potentials were recorded, but generally the repolarization contour was almost linear with time. Contractions were evoked by action potentials but not by the slow, rhythmic fluctuations in the membrane potential.No particular region or structure in the hindgut showed an exclusive pacemaker function. However, there was an evident gradient of increased excitability progressing in an caudal direction from the ileum.In a sodium-free saline, the amplitude of action potentials was remarkable enhanced from 5 to 10 min after the initial change. Even after a 20 min exposure, action potentials were still often present although their frequency and amplitude dropped. Tetrodotoxin (10^–6g/ml) had no. pronounced effect on frequency or amplitude of action potentials. However, spike potentials ceased within 1.5 min after exposure to a sodium and calcium-free saline. When such preparations were re-exposed to a sodium-free saline containing normal calcium, the action potentials reappeared, suggesting that calcium might be a current-carrying ion. Although action potentials in a calcium-free medium showed variability, we generally saw a marked reduction in amplitude of potentials within 5 min. We further observed that 2 mM manganous ion completely abolished action potentials within 2 min. Thus, it seems likely that sodium is not the sole current-carrying ion in cockroach hindgut muscle.The authors express their indebtedness to Ms. Susan Swann, Mr. Gerald Holt, Mr. David Owens, and Ms. Mary Strand for their competent technical assistance.     

The ionic requirements for the initiation of action potentials in insect muscle fibers

Electrical properties of locust leg muscle fibers were studied by means of intracellular electrodes. In most fibers, a depolarizing current pulse initiated a local response. A delayed decrease in membrane resistance appeared with more than about 10 mv depolarization. In some fibers a regenerative response also was found. Membrane constants were measured, applying the short cable model. The value of the space constant λ was 1.6 mm and the calculated value of R_m was about 1750 ohm cm². Action potentials could be elicited when the bathing fluid contained more than 2–5 mM Ba or Sr. Similar responses were seen with 2 mM Ca in the presence of tetraethylammonium (TEA). The overshoot of these action potentials increased with increasing [Ca⁺⁺]_o, [Sr⁺⁺]_o, or [Ba⁺⁺]_o, the increment for a 10-fold increase being about 29 mv for Ca and Sr and between 40 and 50 mv for Ba. These action potentials were inhibited by Mn ions but were not affected by tetrodotoxin or procaine. In solutions containing Ba or Sr, action potentials generated were suppressed by addition of Ca. The removal of Na ions did not change the configuration of the action potential. The results suggest that an increase in permeability to Ca, Ba, or Sr ions makes a major contribution to the initiation of action potentials in this tissue.     

Comparative pharmacological actions of leucokinins V-VIII on the visceral muscles of Leucophaea maderae

1. Leucokinins V-VIII (Lem-K-V to VIII) did not activate visceral muscles of the cockroach Leucophaea maderae uniformly as a group but rather showed a selective action on the muscles of the hindgut. This organ showed a contractile response to all of the leucokinins at 3 x 10(-10) M that was 2-20% above the mean level of spontaneous activity. The maximum response for each peptide was recorded at 2.1 x 10(-7) M. 2. Both the foregut and the oviduct were 100- to 1000-fold less sensitive than the hindgut, and each of the former required more than 10(-8) M to elicit a detectable excitation. The heart, by comparison, did not respond to any of these peptides. 3. The leucokinins caused a protracted excitation of contractile events in the hindgut that lasted for more than 60 min. Moreover, all four peptides evoked contractions from hindguts after membrane depolarization with 158 mM potassium. These results suggest that nonsynaptic receptors for the peptides exist in visceral muscle.     

Pharmacological actions of a new class of neuropeptides, the leucokinins I-IV, on the visceral muscles of Leucophaea maderae

1. Leucokinins I-IV did not activate visceral muscles uniformly as a class but rather showed a selective action on the muscles of the hindgut. This organ showed a contractile response to all of the leucokinins at 3 x 10(-10) M that was 5-10% above the mean level of spontaneous activity. The maximum response for each peptide was recorded at 2.1 x 10(-7) M. 2. Both the foregut and the oviduct were 100-1000 fold less sensitive than the hindgut, and each of the former organs required more than 10(-8) M to elicit a detectable excitation. The heart, by comparison, failed to give consistent responses with any of the peptides. 3. The leucokinins caused a protracted excitation of contractile events in the hindgut that lasted for more than 60 min. Moreover, all four peptides evoked contractions from hindguts after membrane depolarization with 158 mM potassium. 4. This result shows that nonsynaptic receptors for the peptides exist in visceral muscle. The leucokinins showed no evidence of facilitating the reentry of calcium into calcium depleted hindgut preparations.     

Isolation of proctolin,a myotropic peptide,from Periplaneta americana

The isolation of a myotropic substance, proctolin, associated with the innervation of the viscera of the cockroach, Periplaneta americana, is reported. Proctolin, previously described as a neurotransmitter or neurohormone, is a small basic peptide with an estimated molecular weight of 500 to 700. The proctodeal (hindgut) muscles of P. americana contract at threshold levels of about 0·5 ng/ml, equivalent to about 10⁻⁹ M proctolin. Approximately 180 μg proctolin were isolated from 125 kg of whole cockroaches. The isolated peptide was homogeneous on paper and thin-layer chromatography and on high-voltage paper electrophoresis at pH 3·5 and 6·4.     

Neurohormone D induces ionic current changes in cockroach central neurones

The octapeptide neurohormone D (NHD), a member of the family of adipokinetic hormones (AKH-peptides), increases the frequency of spontaneous activity in dorsal unpaired median (DUM) neurones isolated from the terminal ganglion of the cockroach Periplaneta americana. The increase in spike frequency is accompanied by changes in the shape and the amplitude of the single action potentials, e.g. a more pronounced afterhyperpolarization. Effects of NHD on membrane currents were investigated in these DUM cells with whole-cell voltage-clamp measurements. A voltage-independent Ca²⁺ current flowing at the resting potential (I_Ca,R) was found. NHD, at nanomolar concentrations, enhanced this I_Ca,R in a concentration-dependent manner. 0.1 mM Cd²⁺markedly reduced I_Ca,R and in this case I_Ca,R was hardly potentiated by NHD.In the presence of NHD a fast activating Ca²⁺-dependent K⁺current sensitive to charybdotoxin and to low concentrations of tetraethylammonium was augmented. The enhanced afterhyperpolarization of action potentials can be accounted for by the increase in the Ca²⁺-dependent K⁺ current.The changes of the membrane currents induced by NHD are discussed with respect to further effects on the spike pattern and in relation to the previously described mode of action of AKH-peptides in other preparations.Abbreviations NHD neurohormone D - AKH adipokinetic hormone     

Light- and dark-induced action potentials in Physcomitrella patens

Glass microelectrodes were inserted into Physcomitrella patens gametophyte leaves and action potentials (APs) were recorded in response to sudden illumination as well as after darkening, i.e., when the dark-induced membrane depolarization crossed a threshold. Application of 5 mM La³⁺ (a calcium channel inhibitor), 10 mM TEA⁺ (a potassium channel inhibitor) and increased free Ca²⁺ resulted in a loss of excitability. Lack of Ca²⁺ in the external medium did not prevent APs from occurring. It was concluded that during light- dark-induced excitation of Physcomitrella patens, APs might rely upon calcium influxes from the intracellular compartments. APs were not blocked by the proton pump inhibitors (DES, DCCD), although the resting potential (RP) diminished significantly.Key words: action potential, calcium, moss, Physcomitrella patens, plant     

Changes in membrane potential and resistance caused by transient increase of potassium conductance in the unicellular green alga Eremosphaera viridis

The electrophysiological membrane parameters of the unicellular green alga Eremosphaera viridis were determined using an improved computer-supported single-microelectrode technique. These cells developed an average membrane potential of-150 mV in the light and a specific resistance of 1 Ω m² with an external potassium concentration of 1.1 mM and pH 5.5. In the dark, many cells showed a less polarized potential of 30–40 mV and a smaller membrane resistance. At potassium concentrations in the external medium higher than 1 mM, the membrane potential strongly depends on the external potassium content apart from a small electrogenic component. At concentrations lower than 1 mM K⁺, a dependence of the membrane potential upon external potassium concentrations could not be verified. Inserting the internal ion activities in the Goldmann equation shows that, in this range, the proton conductance seems to be predominant over the potassium conductance. Transient changes in the membrane potential and in the membrane resistance were observed after switching off the light, after addition of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea or N,N′-dicyclohexylcarbodiimide, after a sudden decrease in temperature, and after current pulses. These changes resemble the action potentials (AP) found in other plant cells (Chara, Acetabularia). On average, the AP has a delay period of 5.1 s and a duration of 43.8 s showing a sudden decrease and a slower regeneration. The voltage peak during an AP followed exactly the Nernst potential of potassium over a range of external potassium concentrations from 5 μM to 0.2 M. This is true for depolarization or hyperpolarization, depending on the external K⁺-concentration. Tetraethylammonium-hydrogensulphate, a rather specific inhibitor of K⁺ channels in nervous cells, suppressed the AP. The correlation of the appearance of the AP with a short-term opening of potassium channels in the membrane of Eremosphaera is discussed.     

Ionic analyses and effects of ions on contractions of sponge tissues

Summary Contractions of oscular membranes of several marine sponges in response to mechanical stimuli were recorded in various ionic media. Previously, failure to record action potentials and to stimulate electrically was reported.Contractions persisted when sodium was replaced by lithium or potassium but not when the replacement was by choline or sucrose. Contractions were maintained in sea water in which the only univalent cation was potassium; they also persisted in Na-sea water without potassium.Contractions stopped when either calcium or magnesium was omitted, but addition of an equivalent extra amount of the other divalent cation then restored contractions. Contractions were also maintained when strontium was substituted for calcium and magnesium.Analyses of contractile membranes yielded average values for Naples sponges of Na 375 mM/kg H₂O, K 29.8 mM/kg H₂O and inulin space 48.9% of H₂O; for Woods Hole Microciona in mM/kg H₂O: Na 300, K 66, Ca 8.4, Cl 225, inulin space 40% H₂O. The calculated intracellular concentrations for Microciona in mM/kg H₂O were: Na 216, K 104.5, Ca 7.8, Cl 45 yielding i/o ratios of Na 0.5, K 11.6, Ca 0.84, Cl 0.09.It is concluded that, despite relatively normal ionic ratios, mechanical stimuli activate the contractile system in sponges more directly than in muscle.Dedicated to Prof. H. Autrum on the occasion of his 60th birthday.This work was done in part while the author held a Guggenheim Fellowship. Support from NSF grant G 8795 is acknowledged.Assistance of Donald Job, particularly in making all calcium and magnesium measurements, is gratefully acknowledged.     

Possible role of Ca ions in the resting metabolism of frog sartorius muscle during potassium depolarization

Oxygen consumption and Ca exchangeability at different levels of potassium depolarization were studied in frog sartorius muscle. It was found that the changes in oxygen consumption parallel the changes in Ca exchangeability. Procaine (10^?3 M) and CaCl₂ (2.10?2 M) suppressed both extra oxygen consumption and Ca exchangeability at low values of depolarization. At higher values of depolarization procaine and CaCl₂ differed in their action. Procaine favored inhibition of these processes, CaCl₂ caused their activation. The effects of these compounds was not a result of a change in the membrane potential, since their effect on potassium depolarization was found to be small. Relations between oxygen consumption and Ca exchangeability similar to those observed at potassium depolarization seem to exist under conditions where caffeine was applied. It is proposed that the extra oxygen consumption caused by potassium depolarization or on application of caffeine and unaccompanied by mechanical changes is related to the release of Ca from its bound form. Oxygen consumption in isotonic sucrose solution was also studied, but some different data from the above were obtained.     

Calcitonin receptor 1 (AedaeGPCRCAL1) hindgut expression and direct role in myotropic action in females of the mosquito Aedes aegypti (L.)

In anautogenous mosquitoes such as Aedes aegypti females the calcitonin-like diuretic hormone 31 (DH₃₁) stimulates natriuretic fluid excretion from the Malpighian tubules (MTs) after a blood meal. We previously cloned and functionally characterized AedaeGPCRcal1 from A. aegypti, the ortholog of the Drosophila melanogaster DH₃₁ receptor and immunolocalized it in the MTs. However, localization of the calcitonin receptor-like receptor 1 (GPCRCAL1) in the hindgut of any insect is unknown, and specifically, knowledge on its role in hindgut contraction in response to Aedae-DH₃₁ peptide is lacking. We analyzed the expression of AedaeGPCRCAL1 in hindgut by western blot and immunohistochemisty, and evaluated its role in hindgut contractility by application of Aedae-DH₃₁ before and after receptor RNA interference (RNAi). The receptor was detected as a 73 kDa band in western blots of hindgut and immunofluorescence revealed the receptor was expressed in hindgut circular and longitudinal muscles but not in the hindgut epithelial cells. In vitro, incubation in 1 μM solution of Aedae-DH₃₁ peptide significantly increased the hindgut contraction frequency in normal mosquitoes. Hindguts from females treated with AedaeGPCRcal1 dsRNA and incubated with DH₃₁ showed a reduction of 50% percent in their contraction frequency with respect to controls. These results suggest that DH₃₁ hormone released from the brain post-blood meal has a direct and coordinative action on the excretory system, MTs and hindgut, by which AedaeGPCRCAL1 signaling stimulates MT primary urine secretion and hindgut contraction resulting in rapid postprandial fluid excretion.     

Activation of the fast calcium input in the denervated smooth muscle of the cat nictitating membrane

The excitation and contraction features of innervated and sympathetically denervated smooth muscle strips from cat's nictitating membrane have been studied by single sucrose gap arrangement. Increasing of smooth muscle cells sensitivity to drugs were accompanied by elevation of membrane response and the ability to generation of action potentials. Action potentials have been induced by agonists or high potassium concentration in external solution and spontaneously. In innervated muscle action potentials have been evoked as a result of depolarization by high potassium concentration of TEA blockade of potassium conductance. Induced and spontaneously generated action potentials were blocked by organic and inorganic antagonists of potential dependent Ca++ channels. In Ca-free solution action potentials were absent but might be supported by Ba++. Decrease of Na+ had no effect on smooth muscle excitability. It is supposed that activation of potential depended Ca++ channels in smooth muscle cells with pharmaco-mechanical coupling are under influence of sympathetic nerves.     

Effects of leucomyosuppressin on the excitation-concentration coupling of insect Leucophaea maderae visceral muscle

1. Leucomyosuppressin (LMS) inhibited neurally evoked contractions of the hindgut of the cockroach Leucophaea maderae. The threshold for this inhibition of LMS was in the range of 1 × 10⁻¹⁰ M.2. LMS caused a sharp reduction in both l-glutamate and proctolin induced contractions. Dose-response profiles of the effect of LMS (held constant at 10⁻⁸M) on variable amounts of proctolin showed an inhibitory effect at 10⁻⁹ M proctolin and below, but at 5 × 10⁻⁹ M proctolin and above, LMS caused no inhibition.3. Potassium (158 mM) depolarized hindguts treated with LMS (10⁻⁸ M) showed a marked reduction (76% ± 2.1) in the proctolin (10⁻⁸ M) response.4. When calcium depleted preparations were returned to normal calcium levels (2 mM) in the presence of proctolin (10 ⁻⁸ M) a contraction occurred that was 45% ± 4 of the maximum in normal saline solution. However, LMS (10⁻⁸ M) reduced this response to only 28% ± 2 of the maximum.5. Proctolin (10⁻⁸ M) induced contractions in the presence of the manganous ions (2mM) fell to 63% ± 4 of the maximum but on the addition of LMS (10⁻⁸M), such responses fell to only 16% ± 5 of the maximum.6. These results offer evidence for a non-synaptic site of action for LMS and a perturbation of key calcium dependent events in the excitation-contraction coupling sequence of visceral muscle by this peptide.     

Electrical properties of skeletal muscle fibres of the flour moth larva Ephestia kuehniella

The electrical properties of the ventral longitudinal muscle fibres in the flour moth larva Ephestia kuehniella were investigated at rest and during electrical activity. The membrane resting potential was only partially dependent on the K-concentration gradient across the muscle membrane. The electrical constants λ, τ, R_m, R_i, and C_m were determined according to the equations for ‘short cables’ (Table 1). Current-voltage relationships of the muscle membrane were measured: they revealed anomalous as well as delayed rectification of the membrane. Stimulation of the muscle fibres with intracellular current pulses elicited graded action potentials in most fibres; in some fibres ‘all-or-none’ action potentials were generated. In contrast to graded action potentials these ‘all-or-none’ action potentials were propagated without decrement along the muscle fibre. Indirect stimulation of the muscle fibres resulted in large excitatory junction potentials which generally gave rise to action potentials.     

Nickel Substitution for Calcium in Excitation-Contraction Coupling of Skeletal Muscle

In 1962 Frank (22) reported that the addition of any one of a number of divalent cations, including Ni, to a Ca-free Ringer solution prevented the rapid loss of contractility seen in the absence of external Ca. To investigate further the Ni-Ca substitution, studies were made of ⁴⁵Ca and ⁶³Ni exchange during contraction and at rest using frog striated muscle. In contrast to ⁴⁵Ca, it was found that there is no increase of ⁶³Ni uptake associated with a K contracture of the sartorius muscle. The rates of loss of ⁶³Ni and ⁴⁵Ca from resting toe muscles previously bathed in the respective radioisotopes are not significantly different. Resting and action potentials, after 1 hr in a Ringer solution with Ni replacing Ca, closely resemble these potentials in normal Ca-Ringer's solution. Studies on the syneresis of isolated myofibrils indicate that Ni cannot replace Ca in activating this reaction. It is suggested that Ca is required for at least two steps in E-C coupling: one is the spread of excitation at the sarcolemma and transverse tubular system; the second is the activation of actomyosin ATPase. Conceivably Ni can substitute for Ca in the former but not in the latter.     

The effects of modified ringer's solutions on the membrane potentials of innervated and denervated frog sartorius muscle fibers

Comparison has been made between innervated and chronically denervated frog sartorius muscle fibers for resting potentials and a number of features of the action potential. Muscles were obtained from force-fed frogs maintained at room temperature for periods up to one year, and were studied with intracellular microelectrodes. Denervated muscles increased in sensitivity to acetylcholine by 100–400-fold. Studies were made in normal Ringer's solution, and in media in which concentrations of K⁺, Na⁺, Ca⁺⁺, and Cl? were altered. The only significant differences noted between the denervated and the innervated fibers were a reduction in the maximum rate of fall of the action potential (ca. 20%) and an increase in the fall time of the active membrane potential (ca. 25%). These differences were present in normal Ringer's solution and remained when the bathing medium was modified. The resting membrane potential of denervated and innervated muscles varied with log [K⁺]_o in exactly the same manner, and followed the theoretical relation proposed by Hodgkin (Proc. Roy. Soc., B, 148: 1–37, ′58), with the term representing the ratio of the sodium to potassium permeabilities assigned a value of 0.01. The results suggest that (a) the resting sodium and potassium permeabilities are reduced proportionately after denervation, since it is known that denervated frog muscle has a smaller potassium permeability, and (b) the mechanism controlling the increase in potassium conductance during the action potential is less available after denervation. Data indicate that the system controlling the sodium permeability is capable of activation to the same extent as in innervated muscles. Muslces which had been allowed to reinnervate did not show the differences presented by the denervated muscles. Innervated and denervated muscles did not show any significant changes in maximum rates of rise or fall of the action potential, nor of the active membrane potential amplitude over a 30 mV range of resting membrane potentials, indicating that the sodium and potassium permeability systems are fully available in frog muscle at membrane potentials larger than ?80 mV.     

The role of proctolin and glutamate in the excitation-contraction coupling of insect visceral muscle

Proctolin (1 X 10(-10) to 1 X 10(-9) M) had a minimal effect on the spontaneous and evoked electrical events of the hindgut of the cockroach Leucophea maderae. Spontaneous action potentials and contractile activity stopped when the hindgut was exposed to 2 mM Mn2+. Eighty per cent of the response of the hindgut to glutamate was blocked by manganese, but only 35% of the response to proctolin. Hindguts were responsive to proctolin in a calcium-free medium, but not to glutamate. Moreover, proctolin appeared to facilitate the reentry of calcium after depleted preparations were returned to normal levels of external calcium. The results offer evidence for two calcium transmembrane channels in insect visceral muscle.     

Effects of External Calcium Deprivation on Single Muscle Fibers

Deprivation of external calcium causes sudden potentiation of the twitch response of single muscle fibers. The potentiation was 64 ± 8%. Potentiation is simultaneous with membrane depolarization occurring after Ca⁺⁺ removal. This depolarization amounted to 9 ± 2 mv. Ca⁺⁺ removal also alters the action potential. 3 min after calcium withdrawal, action potential amplitude fell by 36 ± 3 mv; maximum rates of rise and fall of the spike decreased by 55 ± 5 and 63 ± 5% respectively. Changes in shape of the A. P. differ from those seen with other potentiators of the twitch response, such as Zn⁺⁺. After short exposure to calcium-free media, potassium-induced contractures show potentiation of peak tension. The S-shaped curve relating potassium contracture tension to log [K]_o shifts to the left after such treatment. Calcium deprivation also increased the rate of relaxation of the contractures. This effect depends on the duration of calcium deprivation, and is probably related to the effect of calcium lack on the membrane. The change in relaxation occurred immediately after calcium deprivation, and was reversed by sudden readmission of calcium. Relaxation of twitch and tetanus responses also were affected by Ca lack, but not as rapidly as potassium contractures. The results suggest that external calcium is not directly involved in the process responsible for tension development, supporting the view that this process is mediated by translocation of intracellular calcium. The relaxation process, however, appears to be rapidly affected by deprivation of external calcium.     

The Effects of pH on the Ionic and Electrical Properties of the Internodal Cells of Chara australis

The effects of pH on the resting and action potentials and onthe fluxes of potassium, sodium, and chloride across the membranesof internodal cells of Chara australis have been investigated. Experiments were carried out in an artificial pond water (A.P.W.)of standard composition: CaCl₂, 01 mM; KCl, 0.1 mM; NaCl, 1.0mM. The resting potential decreased as the pH was lowered from6.5, being depolarized by about 75 mV at pH 4.5. Measurementsof the ion fluxes as a function of pH suggested that this depolarizationwas caused by an increase in the permeability to sodium anda decrease in permeability to potassium at pH 4.5. Action potentialsof constant peak value can be elicited for some time at pH 4.5,but after 20 min or so the cell becomes refractory. All theseeffects on resting and action potentials are fully reversible.We briefly speculate about the mechanism of these pH effects.